KR20170038826A - Anti-orai1 antibody - Google Patents

Abstract

The present invention is to provide a therapeutic and / or prophylactic agent for rejecting an implant, an immunological disease, an allergic disease, an inflammatory disease, a thrombus, a cancer, etc., targeting human Orai1. The provision of a pharmaceutical composition comprising an antibody that specifically recognizes human Orai1 and has an activity of inhibiting human T cell activation, and the like.

Description

Anti-Orai1 antibody {ANTI-ORAI1 ANTIBODY}

The present invention relates to the acquisition of a more potent active anti-Orai1 antibody for the treatment of disorders and diseases.

Calcium-activated activated calcium (CRAC) channels are a subset of store-sensitive channels (SOCs) that open in response to depletion of calcium stores in intracellular endoplasmic reticulum (SOC), and are specific immune cells, particularly cells of the immune system, including T cells or mast cells , Which is responsible for the influx of extracellular calcium and the activation of cells accompanying it. Inhibitors of CRAC channel activity are well known in the art, and their identification and therapeutic potential are described by Feske et al. (Non-Patent Document 1 - 2).

Orai1 (CRACM1: calcium release-activated calcium modulator 1, membrane-through protein 142A: TMEM142A) is a 4-fold transmembrane protein consisting of 301 amino acid residues that constitutes the hole subunit of the CRAC channel by forming homodimers (Patent Document 1, Non-Patent Document 3 - 5). The human Orai1 gene constitutes the Orai gene family together with human Orai2 and human Orai3 having a homology of 90% or more (Non-Patent Document 6), and in some cases, a heterozygote comprising Orai2 and / or Orai3 protein and Orai1 The possibility of formation of hexamer or hexamer has also been reported (Non-Patent Document 7 - 8). Orai1 is an N-terminal and C-terminal cytoplasmic domain conjugated with STIM-1 (Stromal interaction molecule 1) or STIM-2, a protein that detects calcium store depletion in intracellular ER, Extracellular loop domain consisting of about 20 amino acid residues and a second extracellular loop domain consisting of about 40 amino acid residues (Non-Patent Document 9). The DNA and amino acid sequences of Orai1 are publicly available on public databases and can be referenced by fluid session numbers such as NM_032790 and NP_116179 (NCBI).

The molecular function of Orai1 is related to the activation of the CRAC channel in that the innate functional defect of the human Orai1 gene results in loss of CRAC channel activity, loss of the biological response to the immunogen, and loss of severe immunodeficiency (Non-Patent Document 10-11). Thus, a functional inhibitory antibody targeting an Orai1 molecule may be an inhibitor of CRAC channel activity.

From the information suggesting that inhibitors of CRAC channel activity can be used to treat patients such as immune, allergic, inflammatory diseases, cell or organ transplantation, thrombosis, cancer, etc. (Non-patent document 12) The anti-Orai1 antibody with the aim of inhibiting the function has been attempted and its effect has been examined (Non-Patent Documents 2 to 3 and Patent Documents 13 to 14). Although they exhibit inhibition of the activation of T cells by an antibody alone, their inhibitory activity strength is not yet sufficient, and when they are clinically applied as a biological agent targeting Orai1, a high dose or several doses are necessary, or administration It is considered that there is an unmet medical need in that the method is limited.

International Publication WO07 / 081804 pamphlet International Publication WO11 / 063277 pamphlet International Publication WO13 / 091903 pamphlet

 Feske S, Nature Rev. Immunol. 7, p. 690 - 702 (2007)  Derler I, et al., Expert Opin. Drug Discovery 3 (7), p.787 - 800 (2008)  Prakriya M, et al., Nature 443, p. 230-303 (2006)  Vig M, et al., Science 312, p. 1220-1223 (2006)  Park CY, et al., Cell 136, pp. 876-890 (2008)  Mercer JC, et al., J. Biol. Chem. 281, p.24979 -24990 (2006)  Gwack Y, et al., J. Biol. Chem. 282, p.16232 - 16243 (2007)  Hou X, et al., Science 338, p. 1308-1313 (2012)  Vig M, et al., Curr. Biol. 16, p. 2073-2079 (2006)  Feske S, Nature 441, pp. 179-185 (2006)  McCarlca, et al., J. Allergy Clin. Immunol. 124, p. 1311 - 1318 (2009)  McCarlca, et al., J. Immunol. 185, p. 5845 - 5858 (2010)  Lin F, et al., J. Pharmacol. Exp. Ther. 345, p. 225 to 238 (2013)  Cox JH, et al., PLOS ONE 8 (12), e82944 (2013)

It is an object of the present invention to provide a therapeutic and / or prophylactic agent for rejection of an implant, an immunological disease, an allergic disease, an inflammatory disease, a thrombus, a cancer and the like.

The present inventors have obtained a rat anti-Orai1 antibody for the purpose of searching for a substance having a therapeutic and / or preventive effect on rejection of an implant, immune diseases, allergic diseases, inflammatory diseases, thromboses and cancers. The obtained rat anti-Orai1 antibody was humanized, and the CDRs, frameworks and variable regions of the humanized antibody were changed to complete the present invention.

That is, the present invention includes the following inventions.

(1) A CDRH1 comprising CDRH1, CDRH2 and CDRH3, wherein the heavy chain sequence specifically binds to the amino acid sequence set forth in SEQ ID NO: 2, wherein the CDRH1 comprises the amino acid sequence shown in SEQ ID NO: 102, CDRH2 comprises an amino acid sequence represented by any one of SEQ ID NO: 104, 106, 107, or 108, and the CDRH3 comprises an amino acid sequence represented by SEQ ID NO: 109 or 110; And

Wherein the light chain sequence comprises a variable region having CDRL1, CDRL2 and CDRL3, wherein the CDRL1 comprises an amino acid sequence represented by any one of SEQ ID NOs: 93, 94 or 95, and CDRL2 is an amino acid sequence represented by SEQ ID NOs: 96, 97 or 98 CDRL3 consists of the amino acid sequence shown in SEQ ID NO: 101;

Or an antigen-binding fragment thereof.

(2) the heavy chain sequence comprises a variable region having CDRH1, CDRH2 and CDRH3, the CDRH1 comprises the amino acid sequence shown in SEQ ID NO: 102, the CDRH2 comprises the amino acid sequence shown in SEQ ID NO: 106, CDRH3 comprises the amino acid sequence shown in SEQ ID NO: 110; And

Wherein the light chain sequence comprises a variable region having CDRL1, CDRL2 and CDRL3, the CDRL1 comprises an amino acid sequence represented by SEQ ID NO: 93, the CDRL2 comprises an amino acid sequence represented by SEQ ID NO: 96, Comprising an amino acid sequence represented by SEQ ID NO: 101;

(1) or an antigen-binding fragment thereof.

(3) the heavy chain sequence comprises a variable region having CDRH1, CDRH2 and CDRH3, the CDRH1 comprises the amino acid sequence shown in SEQ ID NO: 102, the CDRH2 comprises the amino acid sequence shown in SEQ ID NO: 106, CDRH3 comprises the amino acid sequence shown in SEQ ID NO: 110; And

Wherein the light chain sequence comprises a variable region having CDRL1, CDRL2 and CDRL3, the CDRL1 comprises an amino acid sequence shown by SEQ ID NO: 94, the CDRL2 comprises an amino acid sequence shown by SEQ ID NO: 97, Comprising an amino acid sequence represented by SEQ ID NO: 101;

(1) or an antigen-binding fragment thereof.

(4) the heavy chain sequence comprises a variable region having CDRH1, CDRH2 and CDRH3, the CDRH1 comprises the amino acid sequence shown in SEQ ID NO: 102, the CDRH2 comprises the amino acid sequence shown in SEQ ID NO: 106, CDRH3 comprises the amino acid sequence shown in SEQ ID NO: 110; And

Wherein the light chain sequence comprises a variable region having CDRL1, CDRL2 and CDRL3, the CDRL1 comprises the amino acid sequence shown by SEQ ID NO: 95, the CDRL2 comprises the amino acid sequence shown by SEQ ID NO: 98, Comprising an amino acid sequence represented by SEQ ID NO: 101;

(1) or an antigen-binding fragment thereof.

(5) the heavy chain sequence comprises a variable region having CDRH1, CDRH2 and CDRH3, the CDRH1 comprises the amino acid sequence shown in SEQ ID NO: 102, the CDRH2 comprises the amino acid sequence shown in SEQ ID NO: 107, CDRH3 comprises the amino acid sequence shown in SEQ ID NO: 110; And

Wherein the light chain sequence comprises a variable region having CDRL1, CDRL2 and CDRL3, the CDRL1 comprises an amino acid sequence represented by SEQ ID NO: 93, the CDRL2 comprises an amino acid sequence represented by SEQ ID NO: 96, Comprising an amino acid sequence represented by SEQ ID NO: 101;

(1) or an antigen-binding fragment thereof.

(6) the heavy chain sequence comprises a variable region having CDRH1, CDRH2 and CDRH3, the CDRH1 comprises the amino acid sequence shown in SEQ ID NO: 102, the CDRH2 comprises the amino acid sequence shown in SEQ ID NO: 108, CDRH3 comprises the amino acid sequence shown in SEQ ID NO: 110; And

Wherein the light chain sequence comprises a variable region having CDRL1, CDRL2 and CDRL3, the CDRL1 comprises an amino acid sequence represented by SEQ ID NO: 93, the CDRL2 comprises an amino acid sequence represented by SEQ ID NO: 96, Comprising an amino acid sequence represented by SEQ ID NO: 101;

(1) or an antigen-binding fragment thereof.

(7) the heavy chain sequence comprises a variable region having CDRH1, CDRH2 and CDRH3, the CDRH1 comprises the amino acid sequence shown in SEQ ID NO: 102, the CDRH2 comprises the amino acid sequence shown in SEQ ID NO: 104, CDRH3 consists of the amino acid sequence shown in SEQ ID NO: 109; And

Wherein the light chain sequence comprises a variable region having CDRL1, CDRL2 and CDRL3, the CDRL1 comprises an amino acid sequence represented by SEQ ID NO: 93, the CDRL2 comprises an amino acid sequence represented by SEQ ID NO: 96, Comprising an amino acid sequence represented by SEQ ID NO: 101;

(1) or an antigen-binding fragment thereof.

(8) a heavy chain variable region sequence consisting of amino acid residues 20 to 136 of the amino acid sequence shown by SEQ ID No: 62 and an amino acid residue consisting of the 21st to 126nd amino acid residues represented by SEQ ID No: 56 (1) or (2), or an antigen-binding fragment thereof.

(9) a heavy chain sequence comprising the amino acid residues 20 to 465 or 20 to 466 of the amino acid sequence shown in SEQ ID No: 62 and the amino acid residues 21 to 234 of the amino acid sequence shown in SEQ ID No: 56 (8) or an antigen-binding fragment thereof.

(10) a light chain sequence comprising the amino acid residues 20 to 465 or 20 to 466 of the amino acid sequence shown in SEQ ID No: 70 and the amino acid residues 21 to 234 of the amino acid sequence shown in SEQ ID No: 56 (8) or an antigen-binding fragment thereof.

(11) a heavy chain variable region sequence consisting of amino acid residues 20 to 136 of the amino acid sequence shown by SEQ ID No: 62 and an amino acid residue consisting of the 21st to 126nd amino acid residues represented by SEQ ID No: 58 (1) or (3), or an antigen-binding fragment thereof.

(12) a light chain sequence comprising the amino acid residues 20 to 465 or 20 to 466 of the amino acid sequence shown in SEQ ID NO: 62 and the amino acid residues 21 to 234 of the amino acid sequence shown in SEQ ID NO: 58 (11) or an antigen-binding fragment thereof.

(13) a heavy chain variable region sequence consisting of amino acid residues 20 to 136 of the amino acid sequence shown by SEQ ID No: 62 and an amino acid residue consisting of the 21st to 126nd amino acid residues represented by SEQ ID No: 60 (1) or (4), or an antigen-binding fragment thereof.

(14) A light chain array comprising the amino acid residues 20 to 465 or 20 to 466 of the amino acid sequence shown in SEQ ID NO: 62 and the amino acid residues 21 to 234 of the amino acid sequence shown in SEQ ID NO: 60 (13) or an antigen-binding fragment thereof.

(15) a light chain variable region array comprising a heavy chain variable region sequence consisting of amino acid residues 20 to 136 of the amino acid sequence shown in SEQ ID NO: 64 and amino acid residues 21 to 126 of the amino acid sequence shown in SEQ ID NO: 56 (1) or (5), or an antigen-binding fragment thereof.

(16) a light chain sequence comprising the amino acid residues 20 to 465 or 20 to 466 of the amino acid sequence shown in SEQ ID NO: 64 and the amino acid residues 21 to 234 of the amino acid sequence shown in SEQ ID NO: 56 (15) or an antigen-binding fragment thereof.

(17) a heavy chain variable region sequence consisting of amino acid residues 20 to 136 of the amino acid sequence shown by SEQ ID NO: 66, and light chain variable region arrays comprising amino acid residues 21 to 126 of the amino acid sequence shown in SEQ ID NO: 56 (1) or (6), or an antigen-binding fragment thereof.

(18) A light chain sequence comprising the amino acid residues 20 to 465 or 20 to 466 of the amino acid sequence shown in SEQ ID No: 66 and the amino acid residues 21 to 234 of the amino acid sequence shown in SEQ ID No: 56 (17) or an antigen-binding fragment thereof.

(19) a heavy chain variable region sequence consisting of amino acid residues 20 to 136 of the amino acid sequence shown by SEQ ID No: 43 and an light chain variable region sequence composed of amino acid residues 21 to 126 of the amino acid sequence shown in SEQ ID No: 56 (1) or (7), or an antigen-binding fragment thereof.

(20) a light chain array comprising an amino acid residue at positions 20 to 465 or 20 to 466 of the amino acid sequence shown by SEQ ID No: 43 and amino acid residues at positions 21 to 235 of the amino acid sequence represented by SEQ ID No: 56 (19) or an antigen-binding fragment thereof.

(21) An antigen-binding fragment of an antibody according to any one of (1) to (20), which is selected from the group consisting of Fab, F (ab ') 2, Fab' and Fv.

(22) The antibody according to any one of (1) to (8), (11), (13), (15), (17) or (19).

(23) The pharmaceutical composition according to any one of (1) to (22), or an antigen-binding fragment thereof.

(24) The medicament according to (23), wherein the medicament according to (23) is characterized in that the medicament according to (23) is an agent for inhibiting implantation of an implant, an immune related disease, an inflammatory disease, an agent for the treatment and / or prevention of cancer, an antiplatelet or anti- Composition.

(25) The rejection of the graft is caused by rejection of organ or tissue transplantation such as heart, kidney, liver, bone marrow, skin, and host-to-graft reaction and transplantation of hematopoietic cells (bone marrow, peripheral blood, cord blood etc.) (24), wherein the medicament is a graft-versus-host disease.

(26) An immune-related disease is selected from the group consisting of connective tissue or musculoskeletal system (arthritic rheumatism, ankylosing spondylitis, systemic erythematosus, scleroderma, bundle myositis, dermatomyositis, etc.), blood system (aplastic anemia, idiopathic thrombocytopenic purpura, , Vasculature (Behcet's disease, Wegener's granulomatosis, etc.), epidermal system (psoriasis, pemphigus, albumin, etc.), digestive system (Crohn's disease, ulcerative colitis etc.), nervous system (multiple sclerosis, myasthenia gravis, ), Endocrine system (type 1 diabetes mellitus, autoimmune thyroiditis, bassedosu disease, Hashimoto disease, etc.), allergic diseases such as atopic dermatitis, asthma, anaphylaxis, anaphylactoid reaction, food allergy, rhinitis, otitis media, Inflammatory diseases such as glomerulonephritis (glomerulonephritis, nephrosis, etc.), inflammatory lung diseases (chronic obstructive pulmonary disease, Inflammatory bowel disease, inflammatory bowel disease, etc.), inflammatory liver disease (autoimmune hepatitis, viral hepatitis, etc.), inflammatory heart disease (myocarditis, ischemic heart disease, atherosclerosis Inflammatory diseases such as inflammatory diseases such as inflammatory skin diseases (such as contact dermatitis and eczema), inflammatory diseases such as trachoma and endophthalmitis, inflammatory central nervous system diseases such as meningitis, cerebrospinal fluid and autoimmune encephalitis, , Osteoarthritis, etc.), systemic inflammation (sepsis, bleeding, hypersensitivity, shock symptoms caused by cancer chemotherapy, etc.), and the like.

(27) Examples in which the treatment and / or prevention of cancer is breast cancer, lung cancer, skin cancer, leukemia, etc., and anti-platelet or anti-thrombotic activity are useful for treatment and / or prevention are myocardial infarction, stroke, ischemic heart disease, thrombosis, Examples of the inhibition of Orai1-expressing cell activation that are helpful in the treatment and / or prevention include, but are not limited to, mast cell leukemia, muscular dystrophy, basophilic leukemia, endometriosis, intestinal aggregation myopathy, stromalocytosis, rheumatoid arthritis, ankylosing spondylitis, (24) above, wherein the pharmaceutical composition is a dermatitis, a dermatitis or the like.

(28) A polynucleotide encoding an antibody according to any one of (1) to (22), or an antigen-binding fragment thereof.

(29) A vector comprising any one of the polynucleotides described in (28).

(30) A transformed host cell comprising any one of the polynucleotides described in (28).

(31) A transformed host cell comprising the vector according to (29).

(32) An antibody according to any one of (1) to (22), which comprises culturing the host cell according to (30) or (32) and purifying the antibody from the culture product, A method of producing a fragment.

(33) An antibody or a fragment thereof, which specifically binds to the amino acid sequence set forth in SEQ ID NO: 2 and has a concentration that inhibits 50% of IL-2 released by PMA and A23187-treated Jurkat cells is 80 ng / An antigen-binding fragment of the antibody.

(34) The antibody or the antigen-binding fragment thereof of (33), wherein the concentration that inhibits 50% inhibition of IL-2 released by PMA and A23187-treated Jurkat cells is 10 ng / ml or less.

(35) It is characterized in that the concentration specifically inhibiting 50% of IL-2 released by PMA and A23187-treated human peripheral blood mononuclear cells (PBMC) is 100 ng / ml or less ≪ / RTI > or an antigen-binding fragment thereof.

(36) The antibody or the antigen-binding fragment thereof of (35), wherein the concentration that inhibits 50% of the amount of IL-2 released by PMA and A23187-treated human PBMC is 20 ng / ml or less.

(37) An antibody or a fragment thereof, which specifically binds to the amino acid sequence set forth in SEQ ID NO: 2 and has a concentration that inhibits 50% of the amount of IFN-γ released by PMA and A23187-treated human PBMC is 800 ng / An antigen-binding fragment of the antibody.

(38) The antibody or the antigen-binding fragment thereof according to (37), wherein the concentration that inhibits 50% of the amount of IFN-γ released by PMA and A23187-treated human PBMC is 40 ng / ml or less.

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to obtain a therapeutic agent and / or prophylactic agent for rejection of an implant, an immune disease, an allergic disease, an inflammatory disease, a thrombus, and a cancer, wherein the activity inhibition of the CRAC channel is a mechanism of action.

Fig. 1 shows that R118 and R198 bind to pcDNA3.1-hOrai1-introduced HEK293T cells in a concentration-dependent manner.
Figure 2 shows that R118 and R198 inhibit the release of IL-2 from PMA and A23187 treated Jurkat cells in an additive concentration-dependent manner.
Fig. 3 shows that cR118 and cR198 bind to pcDNA3.1-hOrai1-introduced HEK293T cells in a concentration-dependent manner.
Figure 4 shows that cR118 and cR198 inhibit the release of IL-2 from PMA and A23187 treated Jurkat cells in an additive concentration-dependent manner.
FIG. 5 shows that the humanized anti-human Orai1 antibodies hR198_H1 / L1, hR198_H2 / L2, hR198_H3 / L3 and hR198_H4 / L4 bind to pcDNA3.1-hOrai1-introduced HEK293T cells in a concentration-dependent manner, as in parent strains cR118 and cR198 FIG.
Figure 6 shows that the humanized anti-human Orai1 antibody inhibits the release of IL-2 from PMA and A23187 treated Jurkat cells in an additive concentration-dependent manner.
Figure 7 shows that the mutated Fab clones LCDR60, LCDR67, LCDR83, CE151, PE057, HCDR046, HCDR047, HEP087, HEP124 and HEP237 obtained by the ribosome display, compared to the hR198_H4 / L4- lt; RTI ID = 0.0 > hOrai1 < / RTI >
8 is a graph showing the results obtained when the affinity-amplifying antibodies hR198_H3 / LG1, hR198_HG1 / LG1, hR198_HG1 / LG2, hR198_HG1 / LG3, hR198_HG2 / LG1 and hR198_HG3 / LG1 were compared with hR198_H3 / L3 and hR198_H4 / lt; RTI ID = 0.0 > hOrai1 < / RTI >
Figure 9 shows that the affinity-matched antibody inhibits the release of IL-2 from PMA and A23187 treated Jurkat cells in an additive concentration-dependent manner.
Fig. 10 shows that 10F8, 14F74, and 17F6 antibodies bind to pcDNA3.1-hOrai1-introduced HEK293T cells.
Fig. 11 shows that hR198_HG1 / LG1, hR198_HG1-LALA / LG1, 2C1.1, and 5H3.1 antibodies bind to pcDNA3.1-hOrai1-introduced HEK293T cells.
Figure 12 shows that the anti-Orai1 monoclonal antibody inhibits the release of IL-2 from Jurkat cells treated with PMA and A23187 in an additive concentration-dependent manner.
Figure 13 shows the half-life inhibitory concentration (IC ₅₀ ) and the 80% inhibitory concentration against IL-2 release from Jurkat cells of hR198_HG1 / LG1, hR198_HG1-LALA / LG1, 2C1.1, 5H3.1, 10F8, 14F74, (IC ₈₀ ).
14 is a diagram showing the nucleotide sequence encoding the light chain variable region R118 and the amino acid sequence of the variable region.
FIG. 15 is a diagram showing the nucleotide sequence encoding the R118 midgut variable region and the amino acid sequence of the variable region.
16 is a diagram showing a nucleotide sequence encoding a chain variable region of R198 and an amino acid sequence of the variable region.
17 is a diagram showing the nucleotide sequence encoding the R198 midgut variable region and the amino acid sequence of the variable region.
18 is a diagram showing the nucleotide sequence encoding the human chimeric luminescent cR118 light chain and the amino acid sequence of its light chain.
19 is a diagram showing the nucleotide sequence encoding the human chimeric luminescent cR198 light chain and the amino acid sequence of its light chain.
FIG. 20 is a diagram showing the nucleotide sequence encoding the human chimeric cR118 heavy chain and the amino acid sequence of the heavy chain.
21 is a diagram showing the nucleotide sequence encoding the human chimeric cR198 midblock and the amino acid sequence of the heavy chain.
22 is a diagram showing the nucleotide sequence encoding the hR198_L1 type light chain and the amino acid sequence of the light chain.
23 is a diagram showing the nucleotide sequence encoding the hR198_L2 type light chain and the amino acid sequence of the light chain.
24 is a diagram showing the nucleotide sequence encoding the hR198_L3 type light chain and the amino acid sequence of the light chain.
25 is a diagram showing the nucleotide sequence encoding the hR198_L4 type light chain and the amino acid sequence of the light chain.
26 is a diagram showing the nucleotide sequence encoding the hR198_H1 type heavy chain and the amino acid sequence of the heavy chain.
27 is a diagram showing the nucleotide sequence encoding the hR198_H2 type heavy chain and the amino acid sequence of the heavy chain.
28 is a diagram showing the nucleotide sequence encoding the hR198_H3 type heavy chain and the amino acid sequence of the heavy chain.
29 is a diagram showing the nucleotide sequence encoding the hR198_H4 type heavy chain and the amino acid sequence of the heavy chain.
30 is a diagram showing the nucleotide sequence encoding the hR198_LG1 type light chain and the amino acid sequence of the light chain.
31 is a diagram showing the nucleotide sequence encoding hR198_LG2 type light chain and the amino acid sequence of its light chain.
32 is a diagram showing the nucleotide sequence encoding the hR198_LG3 type light chain and the amino acid sequence of the light chain.
33 is a diagram showing the nucleotide sequence encoding the hR198_HG1 type heavy chain and the amino acid sequence of the heavy chain.
34 is a diagram showing the nucleotide sequence encoding the hR198_HG2 type heavy chain and the amino acid sequence of the heavy chain.
35 is a diagram showing the nucleotide sequence encoding the hR198_HG3 type heavy chain and the amino acid sequence of the heavy chain.
36 is a diagram showing the nucleotide sequence encoding the hR198_H4-LALA type heavy chain and the amino acid sequence of the heavy chain.
FIG. 37 is a diagram showing the nucleotide sequence encoding the hR198_HG1-LALA type heavy chain and the amino acid sequence of the heavy chain.
FIG. 38 corresponds to CDR sequences included in each light chain of cR118, cR198, hR198_L1 to L4 and hR198_LG1 to Lg3, and each light chain of cR118, cR198, hR198_H1 to H4, hR198_HG1 to HG3, hR198_H4-LALA and hR198_HG1- Fig.
39 is a diagram showing the nucleotide sequence encoding the 2C1.1 antibody heavy chain and the amino acid sequence of the heavy chain.
40 is a diagram showing the nucleotide sequence encoding the 2C1.1 antibody light chain and the amino acid sequence of the light chain.
41 is a diagram showing the nucleotide sequence encoding the 5H3.1 antibody heavy chain and the amino acid sequence of the heavy chain.
42 is a diagram showing the nucleotide sequence encoding the 5H3.1 antibody light chain and the amino acid sequence of the light chain.
43 is a diagram showing the nucleotide sequence encoding the 10F8 antibody heavy chain and the amino acid sequence of the heavy chain.
44 is a diagram showing the nucleotide sequence encoding the 10F8 antibody light chain and the amino acid sequence of its light chain.
45 is a diagram showing the nucleotide sequence encoding the 14F74 antibody heavy chain and the amino acid sequence of the heavy chain.
46 is a diagram showing the nucleotide sequence encoding the 14F74 antibody light chain and the amino acid sequence of its light chain.
47 is a diagram showing the nucleotide sequence encoding the 17F6 antibody heavy chain and the amino acid sequence of the heavy chain.
48 is a diagram showing the nucleotide sequence encoding the 17F6 antibody light chain and the amino acid sequence of its light chain.
49 is a diagram showing the nucleotide sequence encoding the hR198_H0 type heavy chain and the amino acid sequence of the heavy chain.
50 is a diagram showing the nucleotide sequence encoding the hR198_H5 type heavy chain and the amino acid sequence of the heavy chain.
Figure 51 shows that the anti-Orai1 monoclonal antibody inhibits the release of IL-2 from PMA and A23187 treated human PBMC in an additive concentration-dependent manner.
Figure 52 shows the half-life inhibitory concentration (IC ₅₀ ) and 80% inhibitory concentration (IL) for IL-2 release from human PBMCs of hR198_HG1 / LG1, hR198_HG1-LALA / LG1, 2C1.1, 5H3.1, 10F8, 14F74, (IC ₈₀ ).
Figure 53 shows that the anti-Orai1 monoclonal antibody suppresses the release of IFN [gamma] from PMA and A23187 treated human PBMC in an additive concentration-dependent manner.
Figure 54 shows the half-inhibition concentration (IC ₅₀ ) and 80% inhibitory concentration (IC ₅₀ ) for IFN gamma release from human PBMCs of hR198_HG1 / LG1, hR198_HG1-LALA / LG1, 2C1.1, 5H3.1, 10F8, 14F74, ₈₀ ).
Figure 55 is a graph showing that hR198_HG1 / LG1 inhibits weight loss associated with graft-versus-host disease caused by the introduction of human PBMC into severe combined immunodeficient mice.
56 is a graph showing that hR198_HG1 / LG1 inhibits a passive skin anaphylactic reaction induced in a human Orai1 knock mouse.
57 is a graph showing that hR198_HG1 / LG1 is inhibited at each time point of 6 hours (A) 24 hours (B) 48 hours (C) after 6 hours of administration of the antigen, to delayed type hypersensitivity induced in human Orai1 knock mice .
FIG. 58 is a graph showing the viscosities measured at the respective concentrations of hR198_HG1 / LG1, hR198_H3 / LG1, hR198_HG1-LALA / LG1, and 2C1.1 antibody at 90 mg / ml, 120 mg / ml and 150 mg / ml.

In the present specification, the word "gene" includes not only DNA but also mRNA, cDNA, and cRNA.

In the present specification, the word " polynucleotide " is used in the same sense as nucleic acid, and includes DNA, RNA, probe, oligonucleotide, and primer.

In the present specification, " polypeptide " and " protein " are used without distinction.

In the present specification, the term "RNA fraction" refers to a fraction containing RNA.

In the present specification, the term " cell " also includes cells in an animal body and cultured cells.

In the present specification, "Orai1" has the same meaning as Orai1 protein.

The term " antigen-binding fragment of an antibody " in the present specification means a fragment of an antibody having an antigen-binding activity, and includes Fab, F (ab ') 2, Fv, scFv, diabody, Multispecific antibodies formed from antibody fragments, and the like. In addition, Fab ', which is a monovalent fragment of the variable region of the antibody treated with F (ab') 2 under reducing conditions, is also included in the antigen-binding fragment of the antibody. However, it is not limited to these molecules as long as they have binding ability to an antigen. These antigen-binding fragments also include proteins produced in suitable host cells using genetically engineered antibody genes as well as the treatment of full-length molecules of the antibody protein with suitable enzymes.

It is known that there are three complementary determining regions (CDRs) in the heavy chain and light chain of the antibody molecule, respectively. The complementarity determining region is also referred to as a hypervariable region and is a region having a particularly high degree of variability in the primary structure within the variable region of the heavy chain and the light chain of the antibody and is a region in which the variable region of the heavy chain and light chain polypeptide chain And are separated into three points on the differential structure. In the present specification, the complementarity determining region of the heavy chain is referred to as CDRH1, CDRH2, and CDRH3 from the amino terminal side of the heavy chain amino acid sequence, and the complementarity determining region of the light chain is referred to as the light chain amino acid sequence CDRL1, CDRL2 and CDRL3 from the amino terminal side. These sites are close to each other in a three-dimensional structure and determine the specificity for the binding antigen.

In the present invention, " hybridize under stringent conditions " means hybridizing at 68 ° C in a commercially available hybridization solution (Expression Hybridization Solution, CLONTECH) , And hybridization was carried out at 68 ° C in the presence of 0.7-1.0 M NaCl. Then, 0.1 to 2-fold concentration of SSC solution (consisting of 150 mM NaCl and 15 mM sodium citrate at 1-fold concentration of SSC) And hybridization is carried out under conditions that can be identified by washing at 68 ° C or conditions equivalent thereto.

In the present invention, " host-to-graft reaction " refers to an immune hyperactivity state of a blood donor observed after organ transplantation and injury of the transplant organs resulting therefrom.

In the present invention, " graft versus host disease " refers to a condition caused by an immunological attack on a graft by a graft cell after hematopoietic stem cell transplantation.

1. Orai1

Orai1 used in the present invention can be purified and used directly from mammals other than humans and humans (eg, guinea pig, rat, mouse, rabbit, pig, sheep, cow, monkey etc.) or chicken T cells or mast cells Alternatively, a cell membrane fraction of the above-mentioned cells can be prepared and used. Alternatively, Orai1 can be obtained by in vitro synthesis or by genetic manipulation to produce a host cell. Specifically, in gene manipulation, it is possible to synthesize Orai1 cDNA in a vector capable of expressing it, and then synthesize it in a solution containing enzymes, substrates and energetic substances necessary for transcription and translation, or a host cell of another prokaryote or eukaryote By expressing Orai1 by transformation, the protein can be obtained.

The nucleotide sequence of the cDNA of human Orai1 is registered in Gen Bank with accession number NM_032790. The nucleotide sequence of the mouse Orai1 cDNA is registered in Gen Bank as accession number NM_175423. The nucleotide sequence encoding human Orai1 is described in SEQ ID NO: 1 of the sequence listing and the amino acid sequence of human Orai1 is set forth in SEQ ID NO: 2 of the sequence listing. In addition, Orai1 is sometimes referred to as Calcium Release-Activated Calcium Modulator 1 (CRACM1) or Transmembrane Protein 142A (TMEM142A), all of which represent the same molecule.

As the cDNA of Orai1, for example, a polymerase chain reaction (hereinafter referred to as " PCR ") is performed using a primer that specifically amplifies Orai1 cDNA, using a cDNA library of organs expressing Orai1 mRNA as a template, (Saiki, RK, et al., Science, (1988) 239, 487-49).

A polynucleotide that hybridizes with a polynucleotide consisting of a nucleotide sequence complementary to a nucleotide sequence encoding Orai1 of a human or a mouse and a protein having a biological activity equivalent to Orai1 under stringent conditions is also Orai1 Lt; / RTI > cDNA. A polynucleotide encoding a splice variant transcribed from the human or mouse Orai1 locus or a polynucleotide hybridizing to the polynucleotide under stringent conditions thereto and encoding a protein having a biological activity equivalent to that of Orai1 is also a polynucleotide of Orai1 lt; / RTI >

Also, in the amino acid sequence of Orai1 of human or mouse, or the amino acid sequence excluding the signal sequence from these sequences, one, two or three or four or five amino acids are substituted, deleted, or added to form an amino acid sequence Proteins with biological activity equivalent to Orai1 are also included in Orai1. In the amino acid sequence or amino acid sequence encoded by the splice variant transcribed from the human or mouse Orai1 locus, an amino acid sequence in which 1, 2 or 3 or 4 or 5 amino acids are substituted, deleted, or added And a protein having biological activity equivalent to that of Orai1 is also included in Orai1.

2. Preparation of anti-Orai1 antibody

The antibody against Orai1 of the present invention can be obtained by immunizing an animal with an arbitrary polypeptide selected from the amino acid sequence of Orai1 or Orai1 using a conventional method, and collecting and purifying an antibody produced in vivo. The species of Orai1 to be an antigen is not limited to humans, and Orai1 derived from a non-human animal such as a mouse or a rat can be immunized to an animal. In this case, antibodies that can be applied to human diseases can be selected by testing the cross-reactivity of human Orai1 with the antibody binding to the obtained heterologous Orai1. Orai1, which is an antigen, can be obtained by producing an Orai1 gene in a host cell by genetic engineering. Specifically, a vector capable of expressing the Orai1 gene may be prepared, introduced into a host cell, and the gene may be expressed to purify the expressed Orai1.

The antibody against Orai1 of the present invention can also be obtained using a DNA immunoassay. DNA immunization is a technique of inducing immunity to an antigen by introducing an antigen expression plasmid into an animal such as a mouse or rat and expressing the antigen in the individual. Examples of the method of gene introduction include a method of injecting a plasmid directly into a muscle, a method of intravenously injecting a complex such as a plasmid and a liposome or polyethyleneimine, a method of using a viral vector, a method in which a plasmid- And a hydrodynamic method in which a large amount of a plasmid solution is intravenously injected rapidly. As a technique for improving the expression level of the gene introduction method by the root of the expression plasmid, there is known a technique called in-vivo electroporation in which electroporation is applied to the same site after the plasmid is introduced (Aihara H, Miyazaki J. Nat Biotechnol. 1998 Sep; 16 (9): 867-70 or Mir LM, Bureau MF, Gehl J, Rangara R, Rouy D, Caillaud JM, Delaere P, Branellec D, Schwartz B, Scherman D. Proc Natl. Acad Sci US A. 1999 Apr 13; 96 (8): 4262-7). This method improves the expression level further by treating the muscle with hyaluronidase before the end of the plasmid (McMahon JM1, Signori E, Wells KE, Fazio VM, Wells DJ Gene Ther. 2001 Aug; 8 (16): 1264 -70).

(Kohler and Milstein, Nature (1975) 256, p. 495-497, Kennet, R. ed., Monoclonal Antibodies, p. 365-367, Plenum Press, NY (1980)), , A hybridoma can be established by fusing an antibody producing cell that produces an antibody against Orai1 and myeloma cells to obtain a monoclonal antibody. Specific examples of such a method are described in the pamphlets of International Publication No. WO09 / 48072 (published on April 16, 2009) and WO10 / 117011 (published October 14, 2010).

Examples of rat anti-human Orai1 antibodies thus established include R118 antibodies and R198 antibodies. The amino acid sequence of the light chain variable region of the R118 antibody is shown in SEQ ID NO: 11 in the sequence listing, and the sequence of the heavy chain variable region of the R118 antibody is shown in SEQ ID NO: In addition, the amino acid sequence of the light chain variable region of the R198 antibody is shown in SEQ ID NO: 15 of the sequence listing, and the sequence of the heavy chain variable region of the R198 antibody is shown in SEQ ID NO: 17 of the sequence listing.

The antibody of the present invention may be added to the above monoclonal antibody against Orai1, and an artificially modified gene recombinant antibody, for example, Chimeric ) Antibodies, humanized antibodies, human antibodies, and the like. These antibodies can be prepared using known methods.

Examples of the chimeric antibody include a chimeric antibody in which a variable region of an antibody, for example, a mouse or a rat-derived antibody, which is mutually different from the variable region and the normal region of the antibody, is conjugated to a normal region derived from a human (Proc. Natl. Acad. Sci. USA, 81, 6851-6855, (1984)).

The chimeric antibody derived from rat anti-human Orai1 antibody R118 comprises light chain comprising light chain variable region consisting of amino acid residues 21 to 126 of SEQ ID NO: 23 and heavy chain comprising amino acid residues 20 to 136 of SEQ ID NO: 27 And an antibody comprising a heavy chain comprising a variable region. An example of such a chimeric antibody derived from R118 is an antibody composed of a heavy chain comprising a light chain consisting of amino acid residues 21 to 234 of SEQ ID NO: 23 and an amino acid residue from 20 to 466 of SEQ ID NO: 27. In the present specification, the antibody is referred to as "cR118" or "cR118 antibody".

In addition, the chimeric antibody derived from rat anti-human Orai1 antibody R198 comprises a light chain comprising a light chain variable region consisting of amino acid residues 21 to 126 of SEQ ID NO: 25 and a light chain comprising a light chain variable region comprising amino acid residues 20 to 136 of SEQ ID NO: And a heavy chain variable region comprising the heavy chain variable region. An example of such a chimeric antibody derived from R198 is an antibody comprising a light chain consisting of amino acid residues 21 to 234 of SEQ ID NO: 25 and amino acid residues 20 to 466 of SEQ ID NO: 29. In the present specification, the antibody is referred to as " cR198 " or " cR198 antibody ".

The above-described arrangement of the chimeric antibody against Orai1 can be artificially modified for the purpose of lowering the heterologous antigenicity to humans, and the human recombinant antibody, humanized, can be produced. The antibody of the present invention includes an antibody that has modified the CDR of the humanized antibody. These antibodies can be prepared using known methods.

Examples of the humanized antibody include an antibody (see Nature (1986) 321, p. 522-525) in which only a complementarity determining region (CDR) is assembled to a human-derived antibody, an antibody The residue may also be an antibody transplanted into a human antibody (WO90 / 07861 pamphlet).

The cR118 and cR198 antibody-derived humanized antibodies retain the CDR sequences of all six species derived from cR118 or cR198 and have an activity of inhibiting the activation of T cells. The light chain variable region of the above humanized antibody is either CDRL1 (RASQSIGNSLS) consisting of the amino acid sequence shown in SEQ ID NO: 92 or CDRL1 (RASQSISNSLS) consisting of the amino acid sequence shown in SEQ ID NO: 116, CDRL2 (STSTLES) comprising the amino acid sequence and CDRL3 (LQFATFPDT) consisting of the amino acid sequence shown by SEQ ID NO: 99, or CDRL3 (LQFATYPDT) shown by SEQ ID NO: 100. The heavy chain variable region of the humanized antibody is composed of any one of CDRH1 (AYYIS) consisting of the amino acid sequence shown in SEQ ID NO: 102 or CDRH1 (SYYIS) shown in SEQ ID NO: 103, CDRH2 YIDMGNGRTNYNARFKG) shown in SEQ ID NO: 105, or CDRH3 (DSNWGVDY) consisting of any one of CDRH2 (YVDMGNGRTNYNEKFKG) shown in SEQ ID NO: 105 and amino acid sequence shown in SEQ ID NO: 109. The amino acid sequence of the CDRs is also shown in Fig.

Preferred examples of the antibody having a combination of CDRs include CDRL1 comprising the amino acid sequence shown in SEQ ID NO: 92, CDRL2 consisting of the amino acid sequence shown in SEQ ID NO: 96, CDRL3 consisting of the amino acid sequence shown in SEQ ID NO: 100, CDRH1 comprising SEQ ID NO: 105, CDRH2 comprising the amino acid sequence shown by SEQ ID NO: 105, and CDRH3 comprising the amino acid sequence shown by SEQ ID NO: 109, CDRL1 comprising the amino acid sequence shown by SEQ ID NO: 92, CDRL3 comprising the amino acid sequence shown by SEQ ID NO: 99, CDRH1 consisting of the amino acid sequence shown by SEQ ID NO: 102, CDRH2 comprising the amino acid sequence shown by SEQ ID NO: 104, and SEQ ID NO: By indicating the amino acid sequence may be an antibody comprising a CDRH3 comprising.

Preferred examples of the above humanized antibody include a light chain comprising a light chain variable region consisting of the amino acid residues 21 to 126 of SEQ ID NO: 31 and a light chain variable region comprising the light chain variable region comprising the amino acid residues 20 to 136 of SEQ ID NO: , A light chain comprising a light chain variable region comprising the amino acid residues 21 to 126 of SEQ ID NO: 33, and a heavy chain variable region comprising the amino acid residues 20 to 136 of SEQ ID NO: 41 Comprising a heavy chain variable region comprising a light chain variable region consisting of amino acid residues 21 to 126 of SEQ ID NO: 35 and an amino acid residue consisting of amino acid residues 20 to 136 of SEQ ID NO: 43, Lt; / RTI > of SEQ ID NO: 37, and antibodies 21 to 126 It may be made of a heavy slow antibody comprising a heavy chain variable region comprising the light chain and 20 to 136 amino acid residues of of SEQ ID NO: 45 comprising a light chain variable region comprising the amino acid residue of a second.

More preferred examples thereof include an antibody consisting of a light chain consisting of amino acid residues 21 to 234 of SEQ ID NO: 31 and an amino acid residue consisting of amino acid residues 20 to 466 of SEQ ID NO: 39, amino acid residues 21 to 234 of SEQ ID NO: 33 An amino acid residue consisting of amino acid residues 20 to 466 of SEQ ID NO: 41, light chain consisting of amino acid residues 21 to 234 of SEQ ID NO: 35, amino acid residues 20 to 466 of SEQ ID NO: 43, An antibody consisting of a heavy chain consisting of a heavy chain consisting of an amino acid residue at positions 21 to 234 of SEQ ID NO: 45 and an amino acid residue at positions 20 to 466 of SEQ ID NO: 45.

The antibody of the present invention may be one in which a mutation has been introduced into the above humanized antibody and the binding ability to Orai1 has been updated. Such a technique is called affinity maturation, and a specific method is the ribosome display method. The ribosome display method is a method of isolating a gene sequence of a protein binding to a target molecule by using a ternary complex in which a protein and mRNA as its genetic information are bound through a ribosome (Stafford RL et al. Protein Eng. Des Flood 2014 (4): 97-109.).

By the above method, the light chain variable region of the antibody to which the gene has been subjected to modification is CDRL1 (RASQSIGGSLS) consisting of the amino acid sequence shown by SEQ ID NO: 93, CDRL1 (HASQNIGGSLS) consisting of the amino acid sequence shown by SEQ ID NO: 94, CDRL2 (STSTLES) consisting of the amino acid sequence shown by SEQ ID NO: 96, CDRL2 (LTSTLDW) consisting of the amino acid sequence shown by SEQ ID NO: 97 or CDRL2 (LTSTLDW) consisting of the amino acid sequence shown by SEQ ID NO: And CDRL3 (LQFAIFPDS) consisting of any one of CDRL2 (LTSSLDW) and amino acid sequence shown by SEQ ID NO: 101. The heavy chain variable region of the antibody subjected to the gene modification includes CDRH1 (AYYIS) consisting of the amino acid sequence shown by SEQ ID NO: 102, CDRH2 (YIDMGNGRTNYNARFKG) consisting of the amino acid sequence shown by SEQ ID NO: 104, amino acid sequence shown by SEQ ID NO: 106 CDRH2 (YIDMGNGRTDYNARFKG) consisting of CDRH2 (YIDMGNGRTDYNARFKG) consisting of the amino acid sequence shown in SEQ ID NO: 107 or CDRH2 (YIDMGNGRTDYNGRFKG) consisting of the amino acid sequence shown in SEQ ID NO: And CDRH3 (DSNWGADY) comprising the amino acid sequence shown by SEQ ID NO: 110. The amino acid sequence of the CDRs is also shown in Fig.

Preferred examples of the antibody having a combination of CDRs include CDRL1 comprising the amino acid sequence shown by SEQ ID NO: 93, CDRL2 consisting of the amino acid sequence shown by SEQ ID NO: 96, CDRL3 consisting of the amino acid sequence shown by SEQ ID NO: 101, CDRH1 comprising SEQ ID NO: 106, CDRH2 comprising amino acid sequence SEQ ID NO: 106, and CDRH3 comprising amino acid sequence SEQ ID NO: 110, CDRL1 comprising amino acid sequence SEQ ID NO: 94, CDRL2 comprising the amino acid sequence shown by SEQ ID NO: 101, CDRL3 consisting of the amino acid sequence shown by SEQ ID NO: 101, CDRH1 consisting of the amino acid sequence shown by SEQ ID NO: 102, CDRH2 comprising the amino acid sequence shown by SEQ ID NO: 106, An antibody comprising CDRH3 comprising the amino acid sequence, CDRL1 consisting of the amino acid sequence shown by SEQ ID NO: 95, CDRL2 consisting of the amino acid sequence shown by SEQ ID NO: 98, CDRL3 consisting of the amino acid sequence shown by SEQ ID NO: 101, CDRH1 comprising the amino acid sequence shown by SEQ ID NO: 106, CDRH2 consisting of the amino acid sequence shown by SEQ ID NO: 110, and CDRH3 comprising the amino acid sequence shown by SEQ ID NO: 110; CDRL1 consisting of the amino acid sequence shown by SEQ ID NO: 93; CDRL3 comprising the amino acid sequence shown by SEQ ID NO: 101, CDRH1 consisting of the amino acid sequence shown by SEQ ID NO: 102, CDRH2 comprising the amino acid sequence shown by SEQ ID NO: 107, An antibody comprising CDRH3 comprising the amino acid sequence shown by SEQ ID NO: 110, CDRL1 comprising the amino acid sequence shown in SEQ ID NO: 93, CDRL2 consisting of the amino acid sequence shown in SEQ ID NO: 96, CDRL3 consisting of the amino acid sequence shown in SEQ ID NO: 101, , CDRH2 comprising the amino acid sequence shown by SEQ ID NO: 108, CDRH3 consisting of the amino acid sequence shown by SEQ ID NO: 110, CDRL1 consisting of the amino acid sequence shown by SEQ ID NO: 93, SEQ ID NO: CDRL3 consisting of the amino acid sequence shown in SEQ ID NO: 101, CDRH1 consisting of the amino acid sequence shown in SEQ ID NO: 102, amino acid sequence shown in SEQ ID NO: 104 Made CDRH2, SEQ ID may be an antibody comprising a CDRH3 comprising the amino acid sequence shown by 109.

Preferred examples of the CDR antibody include a light chain comprising a light chain variable region consisting of the amino acid residues 21 to 126 of SEQ ID NO: 56 and a light chain variable region comprising the amino acid residues 20 to 136 of SEQ ID NO: A heavy chain variable region comprising a light chain variable region consisting of amino acid residues 21 to 126 of SEQ ID NO: 58, and a heavy chain variable region comprising an amino acid residue from the 20th to 136th positions of SEQ ID NO: 62 A light chain comprising a light chain variable region consisting of amino acid residues 21 to 126 of SEQ ID NO: 60, and a heavy chain variable region consisting of amino acid residues 20 to 136 of SEQ ID NO: 62 The antibody comprising the antibody of SEQ ID NO: 56, the antibody of the 21st to 126nd amino acids of SEQ ID NO: 56 An amino acid residue having the amino acid residues 21 to 126 of SEQ ID NO: 56, a light chain variable region comprising a light chain variable region comprising an amino acid residue and an amino acid residue having an amino acid residue at positions 20 to 136 of SEQ ID NO: 64, And a heavy chain variable region comprising an amino acid residue at positions 20 to 136 of SEQ ID NO: 66, and a heavy chain variable region comprising an amino acid residue at positions 21 to 126 of SEQ ID NO: 56 And a light chain variable region comprising the light chain variable region consisting of the amino acid residues 20 to 136 of SEQ ID NO: 43, and a heavy chain variable region comprising the amino acid residues 20 to 136 of SEQ ID NO: 43.

Preferred examples of the antibody comprising the light chain variable region and the heavy chain variable region include a light chain comprising the amino acid residues 21 to 234 of SEQ ID NO: 56 and an intermediate chain consisting of the amino acid residues 20 to 466 of SEQ ID NO: An antibody consisting of a heavy chain comprising the amino acid residues 21 to 234 of SEQ ID NO: 58 and an amino acid residue consisting of the amino acid residues 20 to 466 of SEQ ID NO: 62, and an amino acid residue consisting of the amino acid residues 21 to 234 of SEQ ID NO: Light chain and amino acid residues 20 to 466 of SEQ ID NO: 62, light chain of amino acid residues 21 to 234 of SEQ ID NO: 56, and amino acid residues 20 to 466 of SEQ ID NO: 64 Antibody consisting of heavy chain, SEQ ID NO: 56 An antibody consisting of a light chain consisting of amino acid residues 21 to 234 and an amino acid residue consisting of amino acid residues 20 to 466 of SEQ ID NO: 66, a light chain consisting of the amino acid residues 21 to 234 of SEQ ID NO: 56, Of the amino acid residues 20 to 466 of the heavy chain.

In order to avoid cytotoxicity against normal human Orai1 expressing cells, the effector activity of the antibody is preferably low. The effector activity is known to vary depending on the subclass of the antibody. IgG4 has low ADCC and CDC activity, and IgG2 has CDC activity but low ADCC activity. From this characteristic, it is possible to prepare an antibody in which ADCC and CDC activity are reduced by replacing the normal region of IgG1 with the normal region of IgG2 and 4. In addition, it is possible to produce an IgG1 antibody in which ADCC and CDC activity are reduced by replacing the arrangement of a part of the normal region of IgG1 with reference to IgG2, 4. As an example, Marjan Hezareh et. al. According to Journal of Virology, 75 (24): 12161-12168 (2001), when the 234th and 235th leucine residues of IgG1 (EU index by Kabat et al.) Were substituted with alanine residues, ADCC and CDC activity .

An example of the anti-Orai1 antibody produced by the above method is the heavy chain sequence set forth in SEQ ID NO: 68 or 70. The heavy chain described in SEQ ID NO: 70 or 72 can be used as a therapeutic antibody in combination with each light chain sequence described in this specification. Specific examples of the light chain to be combined include the antibody described in SEQ ID NO: 31, 33, 35, 37, 56, 58 or 60. An antibody comprising a light chain and a heavy chain comprising a light chain comprising the amino acid residues 21 to 234 of SEQ ID NO: 56 and a 20 to 466 amino acid residue of SEQ ID NO: 70, And light chain consisting of amino acid residues 21 to 234 of SEQ ID NO: 70 and amino acid residues 20 to 466 of SEQ ID NO: 70, and light chain and sequence number 70 of the amino acid residues 20 to 466 of the heavy chain.

In addition, it is known that the lysine residue at the carboxyl terminus of the heavy chain of antibodies produced in mammalian cultured cells is deleted (Journal of Chromatography A, 705: 129-134 (1995)), , The two amino acid residues of glycine and lysine are deleted and the proline residue at the carboxyl terminal is newly amidated (Analytical Biochemistry, 360: 75-83 (2007)). However, deletion and modification of these heavy chain sequences do not affect the antigen binding ability and the effect function of the antibody (such as complement activation or antibody-dependent cytotoxicity). Accordingly, the present invention also includes antibodies obtained by subjecting the above-mentioned formulas to an antibody having a deletion of one or two amino acids at the end of the esrboxylic acid and an amidated fragment thereof (for example, proline And the residue is amidated). However, as long as the antigen-binding ability and the effector function are maintained, the carboxyl terminal of the antibody of the present invention is not limited to the above-mentioned kind. The double sheath constituting the antibody relating to the present invention may be any one of the full length and double sheath selected from the group consisting of the deletion products described above, or a combination of any two of them. The ratio of the amount of the individual entities may be influenced by the kind and culture conditions of the mammalian cultured cells producing the antibody related to the present invention. The main component of the antibody related to the present invention is one of the carboxyl terminal 1 Of the amino acid residues are deleted. That is, a heavy chain consisting of amino acid residues 20 to 465 of each heavy chain sequence shown in SEQ ID NOS: 27, 29, 39, 41, 43, 45, 62, 64, 66, 68, To 464th amino acid residues can also be used as the antibody of the present invention.

The antibody obtained by the above method can evaluate the binding property to an antigen and select a preferable antibody. An example of another indicator when comparing the properties of an antibody is the stability of the antibody. Differential scanning calorimetry (DSC) is a method that can quickly and accurately measure the thermal denaturation center (Tm), which is a desirable index of the relative structural stability of the protein. The difference in thermal stability can be compared by measuring the Tm value using DSC and comparing the values. It has been known that the storage stability of antibodies is correlated with the thermal stability of antibodies (Lori Burton, et al., Pharmaceutical Development and Technology (2007) 12, p. 265-273) , A desired antibody can be selected. Other indicators for selecting antibodies include those with high yields in suitable host cells and low cohesiveness in aqueous solutions. For example, since the antibody having the highest yield can not be said to exhibit the highest thermal stability, it is necessary to comprehensively judge based on the indexes described above to select antibodies most suitable for administration to humans.

It is also known to link a full-length array of heavy and light chains of an antibody using an appropriate linker and to obtain a single chain immunoglobulin (Lee, HS, et al., Molecular Immunology (1999) 36, p. 61-71; Shirrmann, T. et al., MAbs (2010), 2, (1) p.1-4). Such a single-chain immunoglobulin is dimerized, so that it is possible to maintain the structure and activity similar to those of an antibody which is originally a tetramer. In addition, the antibody of the present invention may be an antibody having a single heavy chain variable region and no light chain sequence. Such an antibody is referred to as a single domain antibody (sdAb) or a nanobody, and is actually observed in camel or llama and has been reported to retain antigen binding ability (Muyldemans S. et. Al , Protein Eng. (1994) 7 (9), 1129-35, Hamers-Casterman C. et al., Nature (1993) 363 (6428) 446-8). The above antibody can also be interpreted as a kind of antigen-binding fragment of the antibody of the present invention.

It is also possible to enhance the antibody-dependent cytotoxic activity by controlling the sugar chain binding to the antibody of the present invention. WO99 / 54342, WO00 / 61739, WO02 / 31140, and the like are known as techniques for controlling the sugar chain modification of antibodies, but the present invention is not limited thereto.

When an antibody gene is firstly isolated and then introduced into a suitable host, an appropriate host and an expression vector may be used in combination. Specific examples of the antibody gene include a gene encoding a heavy chain sequence of the antibody described in the present specification, and a gene encoding a light chain sequence. When the host cell is transformed, the heavy chain variable gene and the light chain variable gene can be inserted into the same expression vector, or they can be inserted into another expression vector. When eukaryotic cells are used as hosts, animal cells, plant cells, and eukaryotic microorganisms can be used. Examples of the animal cells include (1) mammalian cells such as COS cells (Gluzman, Y. Cell (1981) 23, p.175-182, ATCC CRL-1650) which are cells of monkeys, mouse neonatal cells NIH3T3 USA) or the dehydrofolate reductase deficient strain of Chinese hamster ovary cells (CHO cell, ATCC CCL-61) (ATCC No. CRL-1658) ) 77, p.4126-4220). In the case of using prokaryotic cells, for example, Escherichia coli and Bacillus subtilis may be mentioned. Antibodies are obtained by introducing the desired antibody genes into these cells by transformation, and culturing the transformed cells in vitro. In the above-mentioned culture method, the yield may differ depending on the arrangement of the antibodies, and it is possible to select those antibodies that have equivalent binding activity and whose yield is easy to produce as an indicator.

IgG (IgG1, IgG2, IgG3, IgG4), IgM, IgA (IgA1, IgA2), IgD or IgE and the like can be exemplified, but preferably IgG or IgM , More preferably IgG1 or IgG2.

The antibody of the present invention may also be an antigen-binding fragment of an antibody having an antigen-binding portion of the antibody or a plant thereof. A fragment of the antibody can be obtained by treating the antibody with a protease such as papain or pepsin or by expressing the antibody gene in a suitable culture cell by converting it by a genetic engineering technique. Among such antibody fragments, a fragment retaining all or a part of the functions of the antibody full-length molecule may be referred to as an antigen-binding fragment of the antibody.

Examples of the function of the antibody are generally an antigen binding activity, an activity of neutralizing the activity of the antigen, an activity of enhancing the activity of the antigen, an antibody-dependent cytotoxic activity, a complement dependent cytotoxic activity and a complement dependent cytotoxic activity . The function retained by the antigen-binding fragment of the antibody in the present invention is a binding activity to Orai1, preferably an activity to inhibit activation of T cells, more preferably an activity of IL-2 and / Or inhibiting the production of interferon gamma.

For example, fragments of the antibody include Fab, F (ab ') 2, Fv, or single chain Fv (scFv), diabody (diabodies), linear antibodies, And multispecific antibodies formed from antibody fragments. Fab ', which is a monovalent fragment of the variable region of the antibody treated with F (ab') 2 under reducing conditions, is also included in the fragment of the antibody.

In addition, the antibody of the present invention may be a multispecific antibody having specificity for at least two different antigens. Typically, such a molecule binds to two kinds of antigens (i.e., a bispecific antibody), but the " multispecific antibody "Lt; RTI ID = 0.0 > a < / RTI > specificity.

The multispecific antibody of the present invention may be an antibody of full length or a fragment thereof (for example, F (ab ') 2 bispecific antibody). Bispecific antibodies can be produced by binding heavy chains and light chains (HL pairs) of two kinds of antibodies, or by fusing hybridomas that produce different monoclonal antibodies to produce bispecific antibody-producing fusion cells (Millstein et al., Nature (1983) 305, p. 537-539).

The antibody of the present invention may be a single chain antibody (also referred to as scFv). One chain antibody is obtained by linking the heavy chain variable region and the light chain variable region of the antibody to the linker of the polypeptide (Pluckthun, The Pharmacology of Monoclonal Antibodies, 113 (Rosenberg and Moore, Springer Verlag, New York, p BiscFv fragments produced by linking two scFvs with a polypeptide linker can also be used as bispecific antibodies.

Methods for making single chain antibodies are well known in the art (see, for example, U.S. Pat. No. 4,946,778, U.S. Pat. No. 5,260,203, U.S. Pat. No. 5,091,513, U.S. Pat. No. 5,455,030, and the like). In this scFv, the heavy chain variable region and light chain variable region are linked via a linker, preferably a polypeptide linker, such as not creating a conjugate (Huston, JS et al., Proc. Natl. Acad. Sci. USA (1988), 85, p.5879-5883). The heavy chain variable region and light chain variable region in scFv may be derived from the same antibody or may be derived from another antibody. As the polypeptide linker connecting the variable region, for example, any one-chain peptide consisting of 12 to 19 residues is used.

The DNA encoding the scFv may be a DNA fragment encoding the full-length or heavy chain variable region of the antibody and a DNA portion encoding all or a desired amino acid sequence of the DNA encoding the light chain or light chain variable region A DNA encoding a polypeptide linker portion, and a primer for defining a region in which both ends of the DNA are linked to the heavy chain and the light chain, respectively, are amplified by the PCR method using a pair of primers defining both ends of the template, And amplifying the amplified product.

Once the DNA encoding the scFv is produced, an expression vector containing the same and a host transformed with the expression vector can be obtained according to a conventional method, and further, by using the host, Lt; RTI ID = 0.0 > scFv < / RTI > These antibody fragments can be produced by a host by obtaining and expressing the gene in the same manner as described above.

The antibody of the present invention may be increased in affinity to the antigen. The antibody to be mass-produced may be one kind of antibody or a plurality of antibodies recognizing a plurality of epitopes of the same antigen. Methods for massing the antibody include binding of IgG CH3 domain to two scFvs, binding to streptavidin, and introduction of helix-turn-helix motifs.

The antibody of the present invention may be a polyclonal antibody, which is a mixture of plural kinds of anti-Orai1 antibodies having different amino acid sequences. As an example of the polyclonal antibody, there can be mentioned a mixture of plural kinds of antibodies having different CDRs. As such a polyclonal antibody, a mixture of cells producing different antibodies can be cultured, and purified antibodies can be used from the culture (see WO2004 / 061104).

Antibodies As the water plants, antibodies conjugated to various molecules such as polyethylene glycol (PEG) may be used.

The antibody of the present invention may further be an immunoconjugate in which these antibodies and other agents form a conjugate. An example of such an antibody is that the antibody binds to a radioactive substance or a compound having a pharmacological action (Nature Biotechnology (2005) 23, p.1137-1146).

The obtained antibody can be purified to homogeneity. The separation and purification of the antibody may be carried out by a separation and purification method used in a conventional protein. For example, the antibody can be isolated and purified by appropriately selecting and combining column chromatography, filter filtration, ultrafiltration, salting out, dialysis, polyacrylamide gel electrophoresis for preparation, and isoelectric electrophoresis for preparation (Strategies for Protein Ed Harlow and David Lane, Cold Spring Harbor Laboratory (1988), Antibodies: A Laboratory Manual, Ed., Harling and David Lane, .

Examples of the chromatography include affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration chromatography, reverse phase chromatography, adsorption chromatography, and the like. These chromatographies can be carried out using liquid chromatography such as HPLC or FPLC. Examples of the column used for affinity chromatography include Protein A column and Protein G column. For example, as a column using a Protein A column, Hyper D, POROS, Sepharose F.F. (Pharmacia). It is also possible to purify the antibody by using the carrier immobilized with the antigen and using the binding property to the antigen.

3. Medicines containing the anti-Orai1 antibody

Quot; 2. Among the anti-Orai1 antibodies obtained by the method described in " Preparation of anti-Orai1 antibody ", antibodies capable of neutralizing the biological activity of Orai1 can be obtained. Antibodies that neutralize the biological activity of these Orai1 can inhibit the biological activity of Orai1 in vivo, that is, the activation of the calcium release-dependent calcium channel (CRAC channel) of Orai1-expressing cells, such as T cells or mast cells In terms of medicine, as a therapeutic and / or prophylactic agent for diseases caused by CRAC channel activation of these cells.

Diseases or conditions resulting from CRAC channel activation include rejection of implants, immune related diseases, allergic diseases, inflammatory diseases, antiplatelet or antithrombotic activity, or cancer.

Examples of treatment and / or prevention of graft rejection include rejection of transplantation of organs or tissues such as heart, kidney, liver, bone marrow, skin, host-to-graft reaction and hematopoietic cells (bone marrow, peripheral blood, Lt; RTI ID = 0.0 > and / or < / RTI > prevention of graft versus host disease.

Examples of immune-related diseases include, but are not limited to, connective tissue or musculoskeletal system (arthritic rheumatism, ankylosing spondylitis, systemic erythematosus, nevus, polymyositis, dermatomyositis, etc.), blood system (aplastic anemia, idiopathic thrombocytopenic purpura, (Such as Crohn's disease and ulcerative colitis), nervous system (multiple sclerosis, myasthenia gravis, etc.), visual system (uveitis), vascular system (Behcet's disease, Wegener's granulomatosis, etc.), epidermal system (psoriasis, , Endocrine system (type 1 diabetes, autoimmune thyroiditis, Bassedou disease, Hashimoto disease, etc.).

Examples of allergic diseases include atopic dermatitis, asthma, anaphylaxis, anaphylactoid-like reaction, food allergy, rhinitis, otitis media, drug reaction, insect phase reaction, plant response, latex allergy, conjunctivitis and urticaria.

Examples of inflammatory diseases include inflammatory diseases such as inflammatory renal diseases (glomerulonephritis, nephrosis, etc.), inflammatory lung diseases (chronic obstructive pulmonary disease, cystic fibrosis, interstitial pneumonia, etc.), inflammatory bowel disease (ulcerative colitis, Inflammatory skin disease (such as contact dermatitis, eczema), inflammatory eye diseases (trachoma, endophthalmitis, etc.), inflammatory diseases such as inflammatory liver disease (autoimmune hepatitis, viral hepatitis, etc.), inflammatory heart disease (myocarditis, ischemic heart disease, atherosclerosis, Inflammatory diseases of the joints (such as meningitis, cerebrospinal fluid, autoimmune encephalitis), inflammatory diseases of the joints (such as arthritis and osteoarthritis), systemic inflammation (sepsis, bleeding, hypersensitivity, .

Examples of the anti-platelet or anti-thrombotic activity that are helpful in the treatment and / or prevention include myocardial infarction, stroke, ischemic heart disease, thrombosis and the like.

Examples of cancer treatment include breast cancer, lung cancer, skin cancer, leukemia, and the like.

In addition, it has been suggested that the pathophysiology involved in the histochemical and basophilic mechanisms, mast cell leukemia, mast cell cytology, basophilic leukemia, endometriosis, and the like, which are caused by genetic hyperactivity of the CRAC channel, Asthma, rheumatoid arthritis, ankylosing spondylitis, atopic dermatitis, and the like can be cured by the present antibody.

Examples of the anti-Orai1 antibody as the above-mentioned medicament include humanized antibodies prepared from R118 antibody and / or R198 and CDR variants thereof.

The neutralizing activity of the biological activity of Orai1 by the anti-Orai1 antibody in vitro can be measured, for example, by the activity of inhibiting the activation of T cells expressing Orai1. For example, anti-Orai1 antibody can be added to Jurkat cells, a human T cell-derived cell line, at various concentrations to measure the inhibitory activity against IL-2 release from Jurkat cells by PMA and A23187 stimulation. In addition, the inhibitory activity against IL-2 and interferon gamma release from PBMC by PMA and A23187 stimulation can be measured by adding anti-Orai1 antibody to human peripheral blood mononuclear cells (PBMC) at various concentrations.

Responsible cells for graft-versus-host disease T cells are experimentally proven facts and are widely recognized. When the transplanted T cells recognize the transplanted organism as a foreign substance, it is possible to inhibit the proliferation of IL-2 by self-proliferation by self-produced interleukin 2 (IL-2) or release of inflammatory cytokines such as interferon gamma (IFN- , Resulting in a systemic immune-inflammatory response, resulting in graft-versus-host disease. Therefore, inhibiting the production of these cytokines is linked to prevention or treatment of graft-versus-host disease, and its inhibitory activity can be used as an index to determine the usefulness of the Orai1 antibody as a therapeutic drug.

As a preferred antibody in the present invention, the antibody that specifically binds to the amino acid sequence of SEQ ID NO: 2 and has a concentration that inhibits 50% of the amount of IL-2 released by Jurkat cells treated with PMA and A23187 is 80 ng / An antigen-binding fragment of an antibody or an antibody thereof. More preferably, the antigen-binding fragment of the antibody or the antibody, which has a concentration of 50 ng / ml inhibiting the amount of IL-2 released by PMA and A23187-treated Jurkat cells is 10 ng / . Also provided is an antigen-binding fragment of an antibody or an antibody thereof, and a concentration that inhibits 80% of IL-2 by 80%, wherein the concentration of IL-2 released by PMA and A23187-treated Jurkat cells is less than or equal to 60000 ng / The antibody or an antigen-binding fragment thereof, which is characterized in that the antibody or antigen-binding fragment thereof is less than or equal to 200 ng / ml, is also included in the antibody of the present invention.

As another preferable antibody of the present invention, the antibody that specifically binds to the amino acid sequence of SEQ ID NO: 2 and has a concentration inhibiting 50% of IL-2 released by PMA and A23187-treated human PBMC is 100 ng / Or an antigen-binding fragment thereof. More preferably, the antibody or antigen-binding fragment of the antibody is characterized in that the concentration that inhibits 50% of the amount of IL-2 released by PMA and A23187-treated human PBMC is 20 ng / ml or less. . Also provided is an antigen-binding fragment of an antibody or an antibody thereof, wherein the concentration that inhibits 80% of IL-2 released by PMA and A23187-treated human PBMC is less than or equal to 17000 ng / An antibody or an antigen-binding fragment of the antibody, which is characterized in that the antibody is less than or equal to 400 ng / ml, is also included in the antibody of the present invention.

As another preferred antibody of the present invention, the antibody that specifically binds to the amino acid sequence set forth in SEQ ID NO: 2 and has a concentration that inhibits 50% of the amount of IFN-γ released by PMA and A23187-treated human PBMC is 800 ng / Or less of the antibody or antigen-binding fragment thereof. More preferably, the antibody or antigen-binding fragment thereof is characterized by having a concentration of 50 ng / ml or less inhibiting the amount of IFN-γ released by PMA and A23187-treated human PBMC. . In addition, the antigen-binding fragment of the antibody or the antibody, which has a concentration inhibiting 80% of IFN-γ released by PMA and A23187-treated human PBMC is 300,000 ng / ml or less, and a concentration inhibiting 80% An antibody or an antigen-binding fragment of the antibody, which is characterized in that the antibody is 2000 ng / ml or less, is also included in the antibody of the present invention.

The therapeutic or prophylactic effect of the anti-Orai1 antibody against graft-versus-host disease in vivo using an experimental animal can be confirmed, for example, by measuring inhibition of weight loss in a human PBMC transplanted mouse graft versus host disease model . That is, the next day after 2.0 Gy of X-ray was irradiated to the severe immunodeficient mouse NOG mouse or NSG mouse, a PBS solution containing 15 to 50 million human PBMCs in 1 ml was injected from the tail vein into 200 μl . The X-ray irradiated mouse group without human PBMC was transplanted into the graft-versus-host group and the human PBMC into which only the base material of the antibody was administered as the graft-versus-host group, and the anti-Orai1 antibody By intravenous or intraperitoneal administration in the tail vein, the inhibitory effect of graft versus host group on weight loss is measured.

The therapeutic or prophylactic effect of the anti-Orai1 antibody against various diseases can also be confirmed by the following in vivo evaluation system using a human Orai1 knock mouse.

The effect on dermatitis can be evaluated by administering the protein antiviral solution in the abdominal cavity or vein of the mouse and further immunizing after 2 weeks and then applying the same protein antigen solution to the ear or the abdomen 3 to 6 times every 3 to 2 weeks The thickness of the auricle, the gross score of the dermatitis of the abdomen, the antibody, cytokine, blood biomarker concentration in blood or tissue, the proliferation of cells obtained from skin, peripheral blood, thymus, spleen, lymph node and bone marrow The activity · cytokine production ability · surface antigen was evaluated by comparing between the anti-Orai1 antibody group and the non-administration group.

The effects on the itching can be obtained by applying the mite antigen cream repeatedly 3 to 6 times every 3 to 2 weeks to the auricle or the abdomen, or applying the allenes once a day to weekly to the auricle, the abdomen or the abdomen, or It is possible to measure the number of laryngocyte counts by a magnet mounted on both the head of a mouse and a behavior measuring device, or to measure the number of laryngeal movements of the skin, peripheral blood · Quantified by examining the pathologic features of spinal cord tissue and evaluated by comparing between the anti-Orai1 antibody group and the non-administered group.

The effect on psoriasis can be evaluated by applying Imiquimod to both sides of the auricle or to one side of the auricle and to the body part with completed hairs, intraperitoneally administering a suspension of Zymosan, or administering a cytokine such as IL-23 to one side Intra-intradermal dermatitis can be induced by the weight and thickness of inflammation site, myeloperoxidase activity of neutrophils infiltrating the site, flow cytometry analysis of infiltrated cells, gene analysis, cytokine concentration, etc. , And evaluated by comparing between the anti-Orai1 antibody group and the non-administration group.

The effect on multiple sclerosis is achieved by mixing the Myelin oligodendrocyte glycoprotein or its partial peptide antigen solution with an equivalent amount of Freund's complete adjuvant solution to emulsify and injecting into the lateral or abdominal cavity of the mouse And then the pertussis toxin aqueous solution was administered to the tail vein, and another several days later, the testicular encephalitis induced by the administration of the pertussis toxin solution to the tail vein was expanded from the proximal tail to the forelimb from the proximal one week to two weeks after the start of the experiment Scored by gross observation of paralytic symptoms, and evaluated by comparing between the anti-Orai1 antibody group and the non-administration group.

The effect on arthritis was evaluated by administering the mixture of small-type II collagen and Freund's complete adjuvant and emulsifying them into the mycorrhizae of the mice and performing the same administration two to three weeks later, and scoring , Proliferation activity of cells obtained from skin, peripheral blood, thymus, spleen, lymph node, and bone marrow, cytokine production ability, surface antigen, etc. in the blood or tissue in an antibody, cytokine, blood biomarker concentration, By comparison between the treatment groups.

The effect on colitis can be confirmed by administering trinitrobenzenesulfonic acid in a mouse intestinal tract for 24 hours or by making a 1 - 10% aqueous solution of sodium dextran sulfate free from water for 4 days to 2 weeks, And CD4 + CD25-CD45RBhi T-cells purified and harvested from the spleen were transplanted into the abdominal cavity of Rag2 - / - mice. The body weight during the observation period, the degree of enlargement of the intestinal tract by autopsy after the test, The number, size and pathological characteristics of the cells, antibody, cytokine, blood biomarker concentration in blood or tissue, proliferative activity of cells obtained from the intestinal, peripheral blood, thymus, spleen, lymph node, bone marrow, cytokine production ability, surface antigen etc. Was evaluated by comparing between the anti-Orai1 antibody group and the non-administration group.

The effect on systemic erythematosus can be assessed by inducing systemic erythematosus-like symptoms by administering egg white albumin or other non-polar antigens to the abdomen or subcutaneously at intervals of 5 to 10 days at a maximum of 15 times, Cells were prepared from transplanted organs such as antibody, cytokine, blood biomarker concentration in blood or tissue, antibody or biomarker concentration in urine, or spleen or lymph node collected after completion of the test, And the like were evaluated by comparing between the anti-Orai1 antibody group and the non-administration group.

The effect on hepatitis can be evaluated by inducing hepatitis by administering D-galactose amine alone or in combination with lipopolysaccharide into the abdominal cavity of mice or by administering concanavalin A alone in the tail vein, The GOT and GPT concentrations in the blood and the histopathological status of hepatocellular carcinoma after one hour to one week of administration of the substance are evaluated by comparing between the anti-Orai1 antibody group and the non-administered group.

The effect on the rate of engraftment or prevention of graft-versus-host disease in bone marrow transplantation can be determined by measuring the number of cells in the bone marrow harvested from the femur or tibia of a human Orai1 knock mouse, X-ray irradiated recipient mouse, 4-16 weeks of body weight change or survival rate, antibody / cytokine / blood biomarker concentration in blood or tissue at the end of the test, intestinal, peripheral blood, thymus, spleen, lymph node, The surface antigens, proliferative activity and cytokine production ability of the cells obtained from bone marrow were evaluated by comparing between the anti-Orai1 antibody group and the non-administration group.

The effect on the engraftment rate of the transplant organ was determined by fixing the skin taken from the root of the host mouse to the skin of the human dorsal skin of the human Orai1 knock exposed skin and exposing 4 to 16 weeks after implantation The engraftment status is scored and evaluated by comparing between the anti-Orai1 antibody group and the non-administration group.

The antibodies neutralizing the biological activity of Orai1 thus obtained are useful as medicines for the prevention or treatment of rejection of implants, immunological diseases, allergic diseases, inflammatory diseases, anti-platelet or anti-thrombotic activity, .

An anti-Orai1 antibody can be used alone or in combination with at least one other antibody for the treatment or prophylaxis of cancer, such as rejection of an implant, immune related disease, allergic disease, inflammatory disease, antiplatelet or anti- It may be administered in combination with a therapeutic agent. Other therapeutic agents that can be administered in combination with an anti-Orai1 antibody include folic acid metabolism antagonists, calcineurin inhibitors, adrenocorticosteroid agents, antithymocyte globulin agents, nucleic acid metabolism antagonists, nucleic acid synthesis inhibitors, Or a biological agent targeting a cytokine or cytokine receptor, and the like.

Specific examples of the above therapeutic agents include Methotrexate as a folate metabolic antagonist, Cyclosporin, Taclorims as a calcineurin inhibitor, Methylprednisolone as a corticosteroid steroid, Cyclophosphamide as a nucleic acid synthesis inhibitor, Zetbulin, Lymphoglobuline, Thymoglobulin , Mycophenolate mofetil as a nucleic acid antagonist, and Alemtuzumab, Rituximab, cytokine or cytokine receptor targeting cell surface antigens such as Infliximab, Etanercept, and Rituximab.

Depending on whether they are aimed at rejection of the implant, immune related disease, allergic disease, inflammatory disease, anti-platelet or anti-thrombotic activity, or cancer status or some degree of treatment and / or prevention, Other therapeutic agents may be administered, and other therapeutic agents may be administered simultaneously by being encapsulated in the same formulation. Other therapeutic agents and anti-Orai1 antibodies may be administered simultaneously by being encapsulated in the same formulation. In addition, the anti-Orai1 antibody and other therapeutic agents may be encapsulated in separate preparations and administered simultaneously. In addition, the other therapeutic agent and the anti-Orai1 antibody may be administered separately in succession. That is, after administering another therapeutic agent, a therapeutic agent containing an anti-Orai1 antibody or an antigen-binding fragment of the antibody as an active ingredient is administered, or a therapeutic agent containing an anti-Orai1 antibody or an antigen-binding fragment of the antibody as an active ingredient is administered Other therapeutic agents may be administered after administration. In the case of administration in gene therapy, the gene for the proteinaceous therapeutic agent and the gene for the anti-Orai1 antibody can be inserted downstream of the separate or identical promoter region, and can be introduced into separate or identical vectors.

Targeted drug conjugates: principles and progress ", Advanced Drug Delivery Reviews, (2001) 53, 171-216, may be prepared by conjugating a therapeutic agent to an anti-Orai1 antibody, or fragments thereof, . For this purpose, any antibody fragments can be used as long as they do not completely lose the recognition properties of the T cells in addition to the antibody molecules. For example, fragments of Fab, F (ab ') 2, In the present invention, the same antibody and fragments thereof can be used. The binding pattern of the anti-Orai1 antibody or fragments of the antibody and the therapeutic agent is described in MC Garnet " Targeted drug conjugates: principles and progress ", Advanced Drug Delivery Reviews, (2001) 53, 171-216, GT Hermanson " Bioconjugate Techniques " Academic Press, (1996), Putnam and J. Kopecek, " Polymer Conjugates with Anticancer Activity " Advances in Polymer Science (1995) 122, 55-123. That is, a form in which the anti-Orai1 antibody and the therapeutic agent are chemically bonded directly or via a spacer such as an oligopeptide, or a form in which a therapeutic agent is bound through a suitable drug carrier. Examples of the drug carrier include liposomes and water-soluble polymers. More specifically, the form in which the antibody and the therapeutic agent are contained in the liposome, the liposome and the antibody are bound to each other, and the form in which the therapeutic agent is chemically bonded to the water-soluble polymer (compound having a molecular weight of 1,000 to 100,000) For example, directly or through a spacer such as an oligopeptide, and the antibody is bound to the water-soluble polymer. The binding of antibody (or fragments thereof) to drug carriers such as therapeutic agents, liposomes and water-soluble polymers is described in GT Hermanson Bioconjugate Techniques Academic Press, California (1996), Putnam and J. Kopecek Polymer Conjugates with Anticancer Activity Advances in Polymer Science (1995) 122, 55 - 123, and the like. The inclusion of the therapeutic agent in liposomes can be carried out by methods known to those skilled in the art such as those described in D. D. Lasic, Liposomes: From Physics to Applications, Elsevier Science Publishers BV, Amsterdam (1993) Binding of the therapeutic agent to the water-soluble polymer can be carried out by a method known to a person skilled in the art such as the method described in D. Putnam and J Kopecek "Polymer Conjugates with Anticancer Activity" Advances in Polymer Science (1995) 122, 55-123 . The complex of the antibody (or fragment thereof) and the therapeutic agent for proteinaceous activity (or fragment thereof) can be produced by genetic engineering methods well known in the art, in addition to the above methods.

The present invention also provides a pharmaceutical composition comprising an anti-Orai1 antibody in an amount effective for treatment and / or prevention, and a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative and / or adjuvant.

The present invention relates to a pharmaceutical composition comprising an anti-Orai1 antibody in an amount effective for treatment and / or prevention and at least one therapeutic agent in an amount effective for treatment and / or prophylaxis and a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative and / The present invention also provides a pharmaceutical composition containing the same. Examples of the therapeutic agent include the above-mentioned folic acid metabolism antagonist, calcineurin inhibitor, adrenocorticosteroid agent, antithymocyte globulin agent, nucleic acid metabolism antagonist, nucleic acid synthesis inhibitor, biological agent targeting cell surface antigen, cytokine or cytokine And a biological agent targeting a receptor, but the present invention is not limited thereto.

The substance to be used in the pharmaceutical composition acceptable for the medicinal composition of the present invention is preferably non-toxic to the person to which the medicinal composition is administered, preferably at the dose or the administration concentration.

The pharmaceutical composition of the present invention may contain a substance for preparation to change or maintain the pH, the osmotic pressure, the viscosity, the transparency, the color, the isotonicity, the sterility, the stability, the dissolution rate, the sustained release rate, the absorption rate and the penetration rate. Examples of the substance for the preparation include, but are not limited to, amino acids such as glycine, alanine, glutamine, asparagine, arginine or lysine, antimicrobial agents, antioxidants such as ascorbic acid, sodium sulfate or sodium hydrogen sulfite, , Buffers such as citric acid, boric acid buffer, sodium hydrogencarbonate and Tris-HCl solution; fillers such as mannitol and glycine; chelating agents such as ethylenediamine acetic acid (EDTA); caffeine, polyvinylpyrrolidine, a complexing agent such as β-cyclodextrin or hydroxypropyl-β-cyclodextrin, an extender such as glucose, mannose or dextrin, other carbohydrates such as monosaccharides and disaccharides, a coloring agent, a flavoring agent, a diluent, an emulsifier or polyvinylpyrrolidine Hydrophilic polymers, low molecular weight polypeptides, salt forming counter ions, benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, peptides Preservatives such as glycerin, propylene glycol or polyethylene glycol, sugar alcohols such as mannitol or sorbitol, suspending agents, sorbitan esters, polysorbate 20 A surfactant such as triton, tromethamine, lecithin or cholesterol, a stabilization enhancer such as sucrose or sorbitol, an elasticity enhancer such as sodium chloride, potassium chloride or mannitol or sorbitol , Carriers, excipients, and / or pharmaceutical adjuvants. The amount of the substances for these preparations is preferably 0.01 to 100 times, more preferably 0.1 to 10 times, the weight of the anti-Orai1 antibody. The composition of the preferable pharmaceutical composition in the preparation can be appropriately determined by a person skilled in the art according to the disease to be applied, the route of administration and the like.

The excipient or carrier in the pharmaceutical composition may be either liquid or solid. Suitable excipients and carriers may be water for injection, physiological saline, artificial cerebrospinal fluid, or other materials commonly used in parenteral administration. Neutral saline or physiological saline containing serum albumin may be used as a carrier. The pharmaceutical composition may include Tris buffer at pH 7.0-8.5, acetic acid buffer at pH 4.0-5.5, citrate buffer at pH 3.0-6.2. These buffers may also contain sorbitol or other compounds. The medicinal composition of the present invention includes a medicinal composition comprising an anti-Orai1 antibody, a medicinal composition comprising an anti-Orai1 antibody and at least one therapeutic agent, and the medicinal composition of the present invention is a medicament having a selected composition and required purity, It is prepared as a lyophilized product or liquid. A pharmaceutical composition comprising an anti-Orai1 antibody, and a pharmaceutical composition comprising an anti-Orai1 antibody and at least one anti-bone metabolism therapeutic agent, may be molded as a lyophilized product using a suitable excipient such as sucrose.

The pharmaceutical composition of the present invention may be prepared for parenteral administration or may be prepared for oral absorption of digestive tracts. The composition and concentration of the preparation can be determined according to the administration method. The affinity of the anti-Orai1 antibody against Orai1, that is, the dissociation constant against Orai1 (Kd value) contained in the pharmaceutical composition of the present invention, The higher the Kd value (the lower the Kd value), the better the effect can be exhibited even if the dose is administered to humans. Therefore, the dosage of the pharmaceutical composition of the present invention for humans can be determined based on the results. When the humanized anti-Orai1 antibody is administered to humans, the dosage may be about 0.1 to 100 mg / kg once for 1 to 180 days.

Examples of the form of the pharmaceutical composition of the present invention include injections, drops, suppositories, slivering agents, transdermal absorbers, etc., which include drops.

Although most of the approved antibody formulations are intravenous, subcutaneous administration is preferred in many medical settings. In that case, since the dose is limited to 1.0 to 1.5 ml, a high concentration of antibody solution is required depending on the dosage. However, when the concentration is increased, the viscosity of the chemical liquid is increased. Therefore, there may arise a situation that the injection needle can not be injected because of its high viscosity. In short, in the form of a pharmaceutical composition, when an injection is selected, the property that the viscosity is low has an important meaning to be given priority, and the preferred antibody can be selected from the viscosity as an index.

Example

Hereinafter, the present invention will be described concretely with reference to Examples, but the present invention is not limited thereto. Further, in each of the following examples, each manipulation on gene manipulation was performed in accordance with "Molecular Cloning" (Sambrook, J., Fritsch, EF and Maniatis, T., Cold Spring Harbor Laboratory Press Published in 1989), or when a commercially available reagent or kit is used, it is used according to the instructions of a commercial product.

[Example 1] Production of rat anti-human Orai1 antibody

1) -1 immunity

1) Construction of -1-1 human Orai1 expression vector (pcDNA3.1-hOrai1)

The gene sequence of human Orai1 was purchased from Ultimate ORF clone (Clone No. IOH40869, Life Technologies) cloned in pDONR221 (Life Technologies). The gene sequence is shown in SEQ ID NO: 1 in the sequence listing, and the amino acid sequence in SEQ ID NO: 2 in the sequence listing. In addition, pcDNA3.1-DEST was constructed by transforming pcDNA3.1 (+) into Destination Vector by Gateway Vector Conversion System (Life Technologies). The human Orai1 expression vector pcDNA3.1-hOrai1 was prepared by recombining pcDNA3.1-DEST and att sequences using Gateway LR Clonase Enzyme mix (Life Technologies). The large amount of human Orai1 expression vector was prepared using EndoFree Plasmid Giga Kit (QIAGEN).

1) -1-2 Rat immunization

For immunization, a female of WKY / Izm rat (Nippon Sansei Shisa) was used. First, the rat calf part was pretreated with Hyaluronidase (SIGMA-ALDRICH), and pcDNA3.1-hOrai1 was introduced at the same site. Subsequently, using ECM 830 (BTX), two needle electrodes were used to carry out invisible electroporation on the same site. After repeating the same invivo booster electroporation once every two weeks, the lymph nodes of the rats were collected and used for hybridoma production.

1) -2 Hybridoma production

Lymph node cells and mouse myeloma SP2 / 0-ag14 cells were electrocytically fused using a Hybrimune Hybridoma Production System (Cyto Pulse Sciences) and diluted in Clona Cell-HY Selection Medium D (StemCell Technologies). The monoclonal hybridoma was prepared by recovering the emerging hybridoma colony. Each recovered hybridoma colony was cultured, and screening of anti-human Orai1 antibody-producing hybridomas was carried out using the obtained hybridoma culture supernatant.

1) -3 Primary Screening by Cell-ELISA

1) -3-1 Preparation of antigen-expressing cells for cell-ELISA

HEK293 cells were prepared to a concentration of 7.5 x 10 < ⁵ > cells / ml in DMEM medium containing 10% FBS. On the other hand, pcDNA3.1-hOrai1 or pcDNA3.1-DEST was introduced as a control according to the transfection procedure using Lipofectamine 2000, and 50 μl of each was added to a 96-well plate (Corning) Containing DMEM medium at 37 ° C and 5% CO ₂ for two days. The obtained introduced cells were used in a Cell-ELISA while being adhered.

1) -3-2 Cell-ELISA

Example 1) Introduction of the expression vector prepared in -1-1 After removing the culture supernatant of HEK293 cells, the hybridoma culture supernatant was added to each of the pcDNA3.1-hOrai1 or pcDNA3.1-DEST introduced HEK293 cells, and 4 Lt; 0 > C for 1 hour. The cells in the wells were washed once with PBS containing 5% FBS, and then added with an anti-rat IgG-peroxidase antibody (SIGMA) produced in a rabbit diluted 500-fold with PBS containing 5% FBS And allowed to stand for 1 hour. Cells in the wells were washed six times with PBS containing 5% FBS, and then o-phenylenediamine hydrochloride (OPD solution) (OPD solution (0.05 M trisodium citrate, 0.1 M disodium hydrogenphosphate, Wako Pure Chemical Industries, Ltd.) and H ₂ O ₂ to 0.4 mg / ml and 0.6% (v / v), respectively) were added at 25 μl / well. The color development reaction was stopped by occasionally stirring, and 25 μl / well of 1 M HCl was added to stop the color reaction, and the absorbance at 490 nm was measured with a plate reader (ENVISION: Perkin Elmer). In order to select a hybridoma that produces an antibody specifically binding to human Orai1 expressing on the cell membrane surface, pcDNA3.1-hOrai1 expression vector-introduced HEK293 cells A hybridoma producing a culture supernatant exhibiting a higher absorbance was selected as an anti-human Orai1 antibody positive.

1) -4 Secondary screening by flow cytometry

1) -4-1 Preparation of antigen-expressing cells for flow cytometry analysis

HEK293T cells were seeded in a 225 square cm 2 flask at 5 x 10 ⁴ cells / cm ₂ and cultured overnight in DMEM medium containing 10% FBS at 37 ° C and 5% CO ₂ . The next day, it introduced using Lipofectamine 2000 for pcDNA3.1-DEST respectively in HEK293T cells as pcDNA3.1-hOrai1 and control, which was added to the culture two nights under the conditions of 37 ℃, 5% CO _2. On the next day, the expression vector-introduced HEK293T cells were treated with TrypLE Express (Life Technologies), washed with DMEM containing 10% FBS, and then suspended in PBS containing 5% FBS. The resulting cell suspension was used for flow cytometry analysis.

1) -4-2 Flow cytometry analysis

The binding specificity of the antibody produced by the hybridoma determined to be positive in the Cell-ELISA of Example 1) -3 to human Orai1 was further confirmed by the flow cytometry method. The HEK293T cell suspension prepared in Example 1) -4-1 was centrifuged and the supernatant was removed. The hybridoma culture supernatant was added to each of the pcDNA3.1-DEST-introduced HEK293T cells and the pcDNA3.1-hOrai1-introduced HEK293T cells , And the suspension was allowed to stand at 4 DEG C for 1 hour. After washing once with PBS containing 5% FBS, anti-rat IgG FITC conjugate (SIGMA) diluted 500-fold with PBS containing 5% FBS was added and suspended, and the mixture was allowed to stand at 4 DEG C for 1 hour. After washing three times with PBS containing 5% FBS, the cells were resuspended in PBS containing 5% FBS containing 2 쨉 g / ml 7-aminoactinomycin D (Molecular Probes) (FC500: Beckman Coulter). Data analysis was performed with Flowjo (Tree Star). A histogram of the FITC fluorescence intensity of viable cells was prepared after 7-aminoactothermomycin D positive cells were excluded by gating. The pcDNA3.1-hOrai1 introduction on the fluorescence intensity histogram of HEK293T cells transfected with the control pcDNA3.1-DEST introduced A hybridoma producing a sample in which the histogram of HEK293T cells shifted to the strong fluorescence intensity side was transfected with an anti-human Orai1 antibody- . As a result, 225 hybrid harvesters that were significantly shifted were obtained.

1) -5 isotype determination of antibody

1) -4, R118 and R198, which strongly bind to human Orai1, were selected and identified as antibody isotypes in a hybridoma producing human anti-human Orai1 antibody. Isotype was determined by the Rat monoclonal isotyping test kit (AbD Serotec). As a result, all of the isotypes of rat anti-human Orai1 monoclonal antibodies R118 and R198 were found to be IgG2a, κ chain.

1) -6 Preparation of rat anti-human Orai1 antibody

The rat anti-human Orai1 monoclonal antibody was purified from the hybridoma culture supernatant.

First, R118 and R198-producing hybridomas were grown to a sufficient amount with ClonaCell-HY Selection Medium E, followed by medium exchange with Hybridoma SFM (Life Technologies) supplemented with 20% of Ultra Low IgG FBS (Life Technologies) And cultured for 5 days. The culture supernatant was recovered and sterilized by passing through a 0.45 mu m filter.

The antibody was purified from the above hybridoma supernatant by a one step process of protein G affinity chromatography (4 to 6 ° C). The buffer replacement step after Protein G Affinity Chromatography purification was carried out at 4 to 6 캜. First, the culture supernatant of the hybridoma was applied to a column filled with Protein G (GE Healthcare Bioscience) equilibrated with PBS. After the culture supernatant was put in the column, the column was washed with PBS at a volume of 2 times or more of the column capacity. Next, elution was carried out with a 0.1 M glycine / hydrochloric acid aqueous solution (pH 2.7), and fractions containing the antibody were collected. After adding 1 M Tris-HCl (pH 9.0) to the collected fractions, the pH was adjusted to 7.0 to 7.5, and then buffer substitution with PBS was performed with Centrifugal UF Filter Device VIVASPIN20 (fraction molecular weight UF30K, Sartorius, And concentration was carried out to prepare an antibody concentration of 0.2 mg / ml or more. Finally, the sample was filtered with a Minisart-Plus filter (Sartorius) to obtain a purified sample.

[Example 2] In vitro evaluation of rat anti-human Orai1 antibody

2) -1 Flow cytometry assay of rat anti-human Orai1 antibody

In order to evaluate the binding specificity to Orai1, the cell suspension of pcDNA3.1-DEST-introduced HEK293T or pcDNA3.1-hOrai1-introduced HEK293T cells prepared by the method shown in 1) -4-1 was centrifuged and the supernatant was removed , R118, R198 or Rat IgG control antibody (Beckman Coulter), which was the rat anti-human Orai1 monoclonal antibody prepared in 1) -6, was added thereto, and the mixture was allowed to stand at 4 DEG C for 30 minutes. After rinsing twice with PBS containing 5% FBS, anti-rat IgG FITC conjugate diluted 320-fold with PBS containing 5% FBS was added, suspended, and allowed to stand at 4 캜 for 30 minutes. Washed twice with PBS containing 5% FBS, resuspended in PBS containing 5% FBS containing 1 쨉 g / ml propidium iodide (Life Technologies), and detection was carried out with a flow cytometer (FC500). Data analysis was performed with Flowjo. After the propidium iodide positive cells were excluded from the gates, a histogram of the FITC fluorescence intensity of the live cells was prepared and the average fluorescence intensity (MFI) was calculated. R118 and R198 bind specifically to human Orai1 because they bind to pcDNA3.1-hOrai1-introduced HEK293T cells only in a concentration-dependent manner, as shown in Fig. 1, without binding to pcDNA3.1-DEST introduced HEK293T cells . On the other hand, no binding was observed in the rat IgG control antibody.

2) -2 Rat Anti-Human Orai1 Antibody T Cell Activation Inhibitory Action

Human T cell line, to prepare a Jurkat cell, with 10% FBS, 100 U / ㎖ penicillin and 100 ㎍ / ㎖ streptomycin, 1.5 x 10 ⁶ The concentration of cells / ㎖ in RPMI1640 (Life Technologies, Inc.) containing (Life Technologies, Inc.) And seeded in a 96-well cell culture plate at a rate of 80 쨉 l. 10 μl / well of rat anti-human Orai1 monoclonal antibody R118, R198, or rat IgG control antibody was added thereto and cultured at 37 ° C under 5% CO _{2 for} 60 minutes Respectively. Then, 10 μl / well of 100 ng / ml of PMA (SIGMA) and 1 μg / ml of A23187 (SIGMA) were added (final concentration of 10 ng / ml of PMA and 100 ng / ml of A23187) And cultured at 37 ° C and 5% CO _{2 for} about 16 hours. After the plate was well stirred, the mixture was centrifuged at 600 g for 3 minutes, and the concentration of interleukin-2 (IL-2) contained in the supernatant was measured by ELISA (R & D). Figure 2 shows that the acquired rat anti-human Orai1 monoclonal antibody inhibits the release of IL-2 from PMA and A23187 treated Jurkat cells in an additive concentration-dependent manner. R118 and R198 inhibited the release of IL-2 from Jurkat cells in an additive concentration-dependent manner. On the other hand, no inhibition was observed in the rat IgG control antibody.

[Example 3] Determination of the nucleotide sequence of a cDNA encoding a variable region of a rat anti-human Orai1 antibody

3) -1 cDNA synthesis

(50 mM Tris-HCl (pH 7.5), 250 mM LiCl, 5 mM EDTA (pH 8), 0.5% dodecyl sulfate Li (LiDS), 2.5 mM dithiothreitol (DTT) was mixed with oligo dT25-conjugated magnetic beads (Dynabeads mRNA DIRECT Kit, Invitrogen) to bind the mRNA to magnetic beads. Next, the magnetic beads were immersed in a solution for synthesis of cDNA (50 mM Tris-HCl (pH 7.5), 0.1 mM LiCl, 1 mM EDTA, 0.1% LiDS, 0.1% Triton X- (pH 8.3), 75 mM KCl, 3 mM MgCl ₂ , 5 mM DTT, 0.5 mM dNTP, 0.2% Triton X-100 and 1.2 unit RNase inhibitor (Life Technologies), followed by 12 units SuperScript III Reverse Transcriptase (50 mM potassium phosphate, 4 mM MgCl ₂ , 0.5 mM dGTP, 0.2% Triton X-100, 1.2 units (pH 7.0), and the like) RNase inhibitor (Life Technologies)), followed by 3 'tailing reaction with a reaction solution to which 48 unit terminal transferase and recombinant (Roche) were added.

3) -2 amplification and sequencing of the light chain variable region gene fragment in the rat immunoglobulin

The magnetic beads were washed with TE solution (10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.1% Triton X-100) and then subjected to 5'-RACE PCR to remove the rat immunoglobulin heavy chain and light chain gene Amplification was carried out. That is, the magnetic beads were transferred to a PCR reaction solution (0.2 μM primer, 0.2 mM dNTP, 0.25 unit PrimeSTAR HS DNA polymerase (TAKARA)) and subjected to 35 cycles of reaction at 94 ° C. for 30 seconds and 68 ° C. for 90 seconds. The primer set used was as follows.

PCR primer set (for middle class)

5'-GCTAGCGCTACCGGACTCAGATCCCCCCCCCCCCCCN-3 '(Nhe-polyC-S) (SEQ ID NO: 3)

5'-TCACTGAGCTGGTGAGAGTGTAGAGCCC-3 '(rIgγ-AS1) (SEQ ID NO: 4)

5'-TCACCGAGCTGCTGAGGGTGTAGAGCCC-3 '(rIgγ-AS2) (SEQ ID NO: 5)

PCR primer set (for light chain)

5'-GCTAGCGCTACCGGACTCAGATCCCCCCCCCCCCCCCDN-3 '(Nhe-polyC-S) (SEQ ID NO: 6)

5'-TCAGTAACACTGTCCAGGACACCATCTC-3 '(rIgκ-AS) (SEQ ID NO: 7)

Sequence analysis of the base sequence was performed on the fragment amplified by the PCR reaction. The nucleotide sequence of 5'-CTGGCTCAGGGAAATAGCC-3 '(rIgγ-seq) (SEQ ID NO: 8) as sequence primer for heavy head and 5'-TCCAGTTGCTAACTGTTCC-3' (SEQ ID NO: 9) as light chain sequencing primer Respectively. ≪ tb > < TABLE >

Sequence analysis was carried out using a gene array analyzer (" ABI PRISM 3700 DNA Analyzer; Applied Biosystems " or " Applied Biosystems 3730xl Analyzer; Applied Biosystems "). Sequence reactions were performed using a Dye Terminator Cycle Sequencing System with AmpliTaq DNA polymerase Technologies) and GeneAmp 9700 (Applied Biosystems) were used.

The nucleotide sequences encoding the light chain variable regions of the determined R118 and R198 are shown in SEQ ID NO: 10 (light chain of R118 light chain), 12 (R118 heavy chain) SEQ ID NO: 14 (light chain of R198) and 16 Respectively. The amino acid sequences of the variable regions were shown in SEQ ID NO: 11 (light chain of R118 light chain), 13 (R118 heavy chain), SEQ ID NO: 15 (light chain of R198) and 17 (heavy chain of R198) of the sequence listing, respectively. SEQ ID NOs: 10 and 11 are shown in FIG. 14, SEQ ID NOs: 12 and 13 are shown in FIG. 15, SEQ ID NOs: 14 and 15 are shown in FIG. 16, and SEQ ID NOs: 16 and 17 are shown in FIG.

[Example 4] Production of human chimeric anti-human Orai1 antibody

4) -1 Construction of chimeric and humanized light chain expression vector pCMA-LK

A fragment of about 5.4 kb obtained by digesting the plasmid pcDNA 3.3-TOPO / LacZ (Life Technologies) with restriction enzymes XbaI and PmeI, and a fragment encoding a human κ chain secretion signal and human κ chain normal region shown in SEQ ID NO: 18 of the sequence listing The DNA fragments containing the sequences were ligated using the In-Fusion Advantage PCR Cloning Kit (CLONTECH) to produce pcDNA 3.3 / LK.

PCR was performed using pcDNA3.3 / LK as a template and the following primer sets were subjected to phosphorylation and self-ligation of the obtained fragment of about 3.8 kb to give a signal sequence, a cloning site, and a human κ chain normal region downstream of the CMV promoter , Chimeric and humanized antibody light chain expression vector pCMA-LK was constructed.

Primer set

5'-TATACCGTCGACCTCTAGCTAGAGCTTGGC-3 '(SEQ ID NO: 19: primer 3.3-F1)

5'-GCTATGGCAGGGCCTGCCGCCCCGACGTTG-3 '(SEQ ID NO: 20: primer 3.3-R1)

4) -2 Construction of chimeric and humanized IgG1 type heavy chain expression vector pCMA-G1

a DNA fragment obtained by digesting pCMA-LK with XbaI and PmeI to remove the κ chain secretion signal and the human κ chain normal region and the DNA fragment encoding the amino acid sequence of human heavy chain signal sequence and human IgG1 normal region shown in SEQ ID NO: Were combined using the In-Fusion Advantage PCR cloning kit to generate a chimeric and humanized antibody IgG1 type heavy chain expression vector pCMA-G1 (SEQ ID NO: 1) having a signal sequence, a cloning site, and a human IgG1 heavy chain constant region downstream of the CMV promoter .

4) -3 Construction of human chimeric anti-human Orai1 antibody light chain expression vector

4) Construction of -3-1 human chimeric R118 light chain cR118_L expression vector

A DNA fragment containing the variable region of light chain R118 obtained in Example 3) -2 was synthesized (GENEART artificial gene synthesis service). Chimeric and Humanized Antibody Light Chain Expression The DNA fragment containing the variable region of the light chain of R118 was digested with the same restriction enzyme into a DNA fragment of about 3.4 kb obtained by digesting the general vector pCMA-LK with restriction enzymes XbaI and PmeI A DNA fragment of about 0.7 kb was inserted using Ligation High ver. 2 (TOYOBO) to construct a human chimeric R118 light chain (cR118_L) expression vector. The obtained expression vector was named " pCMA-LK / cR118_L ". The nucleotide sequence encoding the human chimeric luminescent cR118 light chain and the amino acid sequence of its light chain are shown in SEQ ID NOs: 22 and 23 (Fig. 18) of the sequence listing, respectively.

4) Construction of -3-2 human chimeric R198 light chain cR198_L expression vector

A DNA fragment containing the variable region of light chain R198 obtained in Example 3) -2 was synthesized (GENEART artificial gene synthesis service). Chimeric and Humanized Antibody Light Chain Expression The DNA fragment containing the variable region of the light chain of R198 was digested with the same restriction enzyme into a DNA fragment of about 3.4 kb obtained by digesting the general vector pCMA-LK with restriction enzymes XbaI and PmeI A DNA fragment of about 0.7 kb was inserted using Ligation High ver. 2 (TOYOBO) to construct a human chimeric R198 light chain (cR198_L) expression vector. The obtained expression vector was named " pCMA-LK / cR198_L ". The nucleotide sequence encoding the human chimeric luminescent cR198 light chain and the amino acid sequence of its light chain are shown in SEQ ID NOs: 24 and 25 (Fig. 19), respectively, in the sequence listing.

4) Construction of human chimeric anti-human Orai1 antibody heavy chain expression vector

4) -4-1 Construction of human chimeric R118 heavy chain cR118_H expression vector

A DNA fragment containing the variable region of R118 midgut obtained in Example 3) -2 was synthesized (GENEART artificial gene synthesis service). Chimeric and humanized IgG1 type A DNA fragment of about 4.5 kb obtained by digesting the expression vector pCMA-G1 with a restriction enzyme BlpI was digested with the same restriction enzyme to obtain a DNA fragment containing the variable region of R118 heavy chain. kb DNA fragment was inserted using Ligation High ver. 2 to construct a human chimeric R118 heavy chain (cR118_H) expression vector. The obtained expression vector was named " pCMA-G1 / cR118_H ". The nucleotide sequence encoding the human chimeric lysate cR118 heavy chain and the amino acid sequence of the heavy chain are shown in SEQ ID NOS: 26 and 27 (FIG. 20), respectively.

4) -4-2 Construction of expression vector for human chimeric R198 heavy chain cR198_H

A DNA fragment containing the variable region of R198 midgut obtained in Example 3) -2 was synthesized (GENEART artificial gene synthesis service). Chimericized and humanized IgG1 type A DNA fragment of about 4.5 kb obtained by digesting the expression vector pCMA-G1 with a restriction enzyme BlpI was digested with the same restriction enzyme to obtain a DNA fragment containing the variable region of R198 midgut. kb DNA fragment was inserted using Ligation High ver. 2 to construct a human chimeric R198 heavy chain (cR198_H) expression vector. The obtained expression vector was named " pCMA-G1 / cR198_H ". The nucleotide sequence encoding the human chimeric lysine cR198 heavy chain and the amino acid sequence of the heavy chain are shown in SEQ ID NOS: 28 and 29 (FIG. 21), respectively.

4) -5 Preparation of human chimeric anti-human Orai1 antibody

4) -5-1 Production of human chimeric anti-human Orai1 antibody

FreeStyle 293F cells (Life Technologies) were subcultured and cultured according to the manual.

10 ⁷ FreeStyle 293F cells (Life Technologies) from a logarithmic growth phase were inoculated into a 30 ml bottle (Thermo Fisher) and diluted with FreeStyle293 expression medium (Life Technologies) to prepare 9 ml. ₂ < / RTI > incubator at 135 rpm for 1 hour. 30 μg of Polyethyleneimine (Polyscience # 24765) was dissolved in 500 μl of Opti-Pro SFM (Life Technologies), followed by incubation with human chimeric anti-human Orai1 antibody prepared using QIAGEN Plasmid Maxi Kit (QIAGEN) (4 ㎍) and human chimeric anti-human Orai1 antibody light chain expression vector (6 ㎍) were suspended in 500 의 of Opti-Pro SFM. To 500 μl of the mixture of polyethyleneimine / Opti-Pro SFM, 500 μl of the expression vector / Opti-Pro SFM mixture was added, carefully stirred, and allowed to stand for another 5 minutes before being added to FreeStyle 293F cells. The culture supernatant obtained by shaking culture at 95 rpm for 5 to 7 days in an 8% CO ₂ incubator at 37 ° C was filtered with a 0.22 μm MILEX filter (Millipore).

The human chimeric R118 obtained by the combination of pCMA-G1 / cR118_H and pCMA-LK / cR118_L was named "cR118". Similarly, the human chimeric R198 obtained by the combination of pCMA-G1 / cR198_H and pCMA-LK / cR198_H was named "cR198".

4) -5-2 Purification of human chimeric anti-human Orai1 antibody

4) The culture supernatant obtained in -5-1 was purified by one step of r-protein A affinity chromatography. First, 10 ml of the culture supernatant was applied to MabSelectSuRe (GE Healthcare Bioscience) equilibrated with PBS. After the culture broke into the column, the column was washed with 7 ml of PBS. The eluate was then eluted with 4 ml of histidine buffer (25 mM Histidine, 5% Sorbitor, pH 6.0) on a PD-10 desalting column (GE Healthcare Bioscience) . Finally, the sample was concentrated to about 100 μl with Amicon Ultracel 30K (cut-off molecular weight 30 K, Millipore) to obtain a purified sample.

[Example 5] In vitro activity of human chimeric anti-human Orai1 antibody

5) -1 flow cytometry, the antigen-binding activity of human chimeric anti-human Orai1 antibody

In order to evaluate the binding specificity to human Orai1, the cell suspension of pcDNA3.1-DEST-introduced HEK293T or pcDNA3.1-hOrai1-introduced HEK293T prepared by the method shown in 1) -4-1 was centrifuged and the supernatant was removed Then, human chimeric anti-human Orai1 antibody cR118, cR198, or a human IgG control antibody (Jackson Immunoresearch), which was prepared at 4) -5, was added to each, and the mixture was allowed to stand at 4 ° C for 30 minutes. After washing twice with PBS containing 5% FBS, an anti-human IgG FITC conjugate (Jackson Immunoresearch) diluted 100-fold with PBS containing 5% FBS was added, and the mixture was suspended at 4 캜 for 30 minutes . The cells were washed twice with PBS containing 5% FBS, resuspended in PBS containing 5% FBS containing 1 쨉 g / ml Propidium iodide (Invitrogen), and detected with flow cytometer (FC500) Respectively. Data analysis was performed with Flowjo. After the propidium iodide positive cells were excluded from the gates, a histogram of the FITC fluorescence intensity of the live cells was prepared and the average fluorescence intensity (MFI) was calculated. As shown in Fig. 3, cR118 and cR198 bind specifically to human Orai1, since they bind to pcDNA3.1-hOrai1-introduced HEK293T cells only in a concentration-dependent manner without binding to pcDNA3.1-DEST-introduced HEK293T cells . On the other hand, no binding was observed in the human IgG control antibody.

5) -2 inhibition of human T cell line activation of human chimeric anti-human Orai1 antibody

Human T cell line, the Jurkat cells, 10% FBS, 100 U / ㎖ penicillin and 100 ㎍ / ㎖ in RPMI1640 containing streptomycin, 1.5 x 10 ⁶ cells / ㎖ was prepared at a concentration of 80 in a 96-well cell culture plate And 10 μl / well of human chimeric anti-human Orai1 antibodies cR198 and cR118 and mother strain R198, R118, or human IgG control antibody was added thereto, and the mixture was pre-treated at 37 ° C. and 5% CO _{2 for} 60 minutes. Thereafter, 10 ng / well of 100 ng / ml PMA and 1 μg / ml of A23187 was added, well stirred, and then cultured at 37 ° C. and 5% CO _{2 for} about 16 hours. After the plate was well stirred, it was centrifuged at 600 g for 3 minutes, and the concentration of IL-2 contained in the supernatant was measured by ELISA. Figure 4 shows that the produced human chimeric anti-human Orai1 antibody suppresses the release of IL-2 from Jurkat cells treated with PMA and A23187 in an additive concentration-dependent manner. cR118 and cR198 inhibited the release of IL-2 from Jurkat cells in an additive concentration-dependent manner, and the inhibition intensity was equivalent to that of parent strain, R198. On the other hand, no inhibition was observed with the human IgG control antibody.

[Example 6] Design of humanized version hR198 of human chimeric anti-human Orai1 antibody cR198

6) -1 Molecular modeling of the variable region of R198

Molecular modeling of the variable region of cR198 was performed by a method generally known as homology modeling (Methods in Enzymology, 203, 121-153, (1991)). The primary sequence of the variable region of human immunoglobulin, which is registered in the Protein Data Bank (Nuc. Acid Res. 28, 235-242 (2000)) (a three dimensional structure derived from the X-ray crystal structure is available ) Was compared with the variable region of R198 determined above. As a result, 1AJ7 was selected as having the highest sequence homology to the light chain variable region of cR198. Also, 1XGY was selected as having the highest sequence homology to the variable region of the heavy chain of cR198. The three-dimensional structure of the framework region was produced by obtaining the " framework model " by combining the coordinates of 1AJ7 and 1XGY corresponding to the light chain and heavy chain of cR198. The CDRs of cR198 are described in Thornton et al. CDRL2, CDRL3, CDRH1, and CDRH2 were assigned to clusters 11A, 7A, 9A, 10A, and 10A, respectively, according to the classification of E. coli (J. Mol. Biol., 263, 800-815, (1996) CDRH3 was classified as k (6) - using the H3 rule (FEBS letter 399, 1-8 (1996)). Subsequently, a representative composition for each CDR was assembled into the framework model.

Finally, energy calculations were performed to exclude adverse interatomic contacts, in order to obtain a molecular model with potential for variable domains of cR198 in terms of energy. The above procedure was carried out using a commercially available protein stereostructure predicting program Prime and a sitting search program MacroModel (Schrodinger, LLC).

6) -2 Design of amino acid sequence for humanized R198

Construction of the humanized R198 antibody was performed by methods generally known as CDR grafting (Proc. Natl. Acad. Sci. USA 86, 10029-10033 (1989)). The acceptor antibody was selected based on amino acid homology in the framework region.

The sequence of the framework region of cR198 was compared to all human frameworks of the Kabat database of antibody amino acid sequences (Nuc. Acid Res. 29, 205-206 (2001)), 0.0 > 71% < / RTI > sequence homology with the region. The amino acid residues of the framework regions for 1C10'CL were aligned with the amino acid residues for cR198 to identify the locations where the different amino acids were used. The location of these residues is analyzed using a three-dimensional model of cR198 constructed above and the donor residues to be grafted onto the acceptor are described by Queen et al. (Proc. Natl. Acad. Sci. USA 86, 10029-10033 (1989)).

By introducing several selected donor residues into the acceptor antibody, the humanized R198 sequence was constructed as described in the Examples below.

Also, the CDR-modified humanized R198 sequence in which one to five amino acid residues of each CDR and FR of cR198 were substituted with the amino acid residues of cR118 was constructed as described in the following examples.

6) -3 Design of Humanized R198 Light Chain hR198_L

6) -3-1 hR198_L1 type light chain:

The threonine residue at amino acid position 30 of the light chain of cR198 shown in SEQ ID NO: 25 in the sequence listing is substituted with serine residue at serine residue at position 32, serine residue at residue 32, valine residue at residue 35, The residue of the 42th serine residue is referred to as a threonine residue, the residue of the 61st aspartic acid residue as a glycine residue, the 62th glycine residue as a lysine residue, the 63rd serine residue as an alanine residue, the 64th valine residue The serine residue at the 92nd position is substituted with a threonine residue, the 94th serine residue with a threonine residue, the 96th threonine residue with a serine residue, the 99st glutamic acid residue with a glutamine residue, As the proline residue, the 120th threonine residue as the glutamine residue, the 124th leucine residue as the valine residue, the 126th leucine residue as the A leucine residue, an arginine residue in the 127th lysine residues, a humanized light chain R198 is designed to involve substituting an alanine residue for the 129th threonine residue with was named as "hR198_L1 type light chain".

The nucleotide sequence encoding the hR198_L1 type light chain is SEQ ID NO: 30 in the sequence listing, the nucleotide numbers 61 to 702 encoding the mature light chain in which the signal sequence is excluded, and the nucleotide numbers 61 to 378 encoding the variable region. The amino acid sequence of the hR198_L1 type light chain is SEQ ID NO: 31, the signal sequence is mature light chain having the amino acid number 21 to 234, and the variable region is the amino acid number 21 to 126. The arrangement of the array Nos. 30 and 31 is also shown in Fig. 22, respectively. Each CDR array and corresponding array number are shown in FIG.

6) -3-2 hR198_L2 type light chain:

The threonine residue at amino acid position 30 of the light chain of cR198 shown in SEQ ID NO: 25 in the sequence listing is substituted with serine residue at serine residue at position 32, serine residue at residue 32, valine residue at residue 35, The serine residue at the 42nd position is substituted with a threonine residue, the 61st aspartic acid residue is substituted with the glycine residue, the 62th glycine residue with the lysine residue, the 63rd serine residue with the alanine residue, As the threonine residue at the 94th position, as a threonine residue at the 94th position, as a serine residue at the 96th position, as a glutamine residue at the 99th position, as a proline residue at the 100th position, As the glutamine residue, the 124th leucine residue as the valine residue, the 126th leucine residue as the isoleucine residue, the 127th arginine residue In a lysine residue, a humanized light chain R198 is designed to involve substituting an alanine residue for the 129th threonine residue with was named as "hR198_L2 type light chain".

The nucleotide sequence encoding the hR198_L2 type light chain is SEQ ID NO: 32 in the sequence listing, the nucleotide numbers 61 to 702 encoding the mature light chain in which the signal sequence is excluded, and the nucleotide numbers 61 to 378 encoding the variable region. The amino acid sequence of the hR198_L2 type light chain is SEQ ID NO: 33, the mature light chain in which the signal sequence is deleted consists of the amino acid numbers 21 to 234, and the variable region is composed of the amino acid numbers 21 to 126 thereof. The arrangement of the array Nos. 32 and 33 is also shown in Fig. 23, respectively. Each CDR array and corresponding array number are shown in FIG.

6) -3-3 hR198_L3 type light chain:

The threonine residue at amino acid position 30 of the light chain of cR198 shown in SEQ ID NO: 25 in the sequence listing is substituted with serine residue at serine residue at position 32, serine residue at residue 32, valine residue at residue 35, The residue of the 42th serine residue is referred to as a threonine residue, the residue of the 61st aspartic acid residue as a glycine residue, the 62th glycine residue as a lysine residue, the 63rd serine residue as an alanine residue, the 64th valine residue The serine residue at the 92nd position is substituted with a threonine residue, the 94th serine residue with a threonine residue, the 96th threonine residue with a serine residue, the 99st glutamic acid residue with a glutamine residue, As the proline residue, the 114th tyrosine residue as the phenylalanine residue, the 120th threonine residue as the glutamine residue, the 124th leucine residue The humanized R198 light chain designed by replacing the 126th leucine residue with the isoleucine residue, the 127th arginine residue with the lysine residue, and the 129th alanine residue with the threonine residue is referred to as " hR198_L3 type light chain Chain ".

The nucleotide sequence encoding the hR198_L3 type light chain is SEQ ID NO: 34 in the sequence listing, the nucleotide numbers 61 to 702 encoding the mature light chain in which the signal sequence is excluded, and the nucleotide numbers 61 to 378 encoding the variable region. The amino acid sequence of hR198_L3 type light chain is SEQ ID NO: 35, the signal sequence is mature light chain having amino acid number 21 to 234, and the variable region is amino acid number 21 to 126. The arrangement of SEQ ID NOS: 34 and 35 is also shown in FIG. 24, respectively. Each CDR array and corresponding array number are shown in FIG.

6) -3-4 hR198_L4 type light chain:

The threonine residue at amino acid position 30 of the light chain of cR198 shown in SEQ ID NO: 25 in the sequence listing is substituted with serine residue at serine residue at position 32, serine residue at residue 32, valine residue at residue 35, The serine residue at the 42nd position is substituted with a threonine residue, the 61st aspartic acid residue is substituted with the glycine residue, the 62th glycine residue with the lysine residue, the 63rd serine residue with the alanine residue, As the threonine residue at the 94th position, as a threonine residue at the 94th position, as a serine residue at the 96th position, as a glutamine residue at the 99th position, as a proline residue at the 100th position, As the glutamine residue, the 124th leucine residue as the valine residue, the 126th leucine residue as the phenylalanine residue, the 120th threonine residue as the glutamine residue, Group was named as a isoleucine residue, an arginine residue 127 of a second lysine residue, the 129th to R198 humanized light chain designed to involve replacing the alanine residue with threonine residues "hR198_L4 type light chain" of.

The nucleotide sequence encoding the hR198_L4 type light chain is SEQ ID NO: 36 in the sequence listing, the nucleotide sequence numbers 61 to 702 are those encoding the mature light chain in which the signal sequence is excluded, and the nucleotide numbers 61 to 378 are those encoding the variable region. The amino acid sequence of the hR198_L4 type light chain is SEQ ID NO: 37 in the sequence listing, the mature light chain in which the signal sequence is excluded is in the amino acid number 21 to 234, and the variable region is in the amino acid number 21 to 126 in the amino acid sequence. The arrangement of array Nos. 36 and 37 is also shown in Fig. 25, respectively. Each CDR array and corresponding array number are shown in FIG.

6) -4 Design of humanized R198 heavy chain hR198_H

6) -4-1 hR198_H1 type double yarn:

The amino acid of the cR198 heavy chain represented by SEQ ID NO: 29 in the sequence listing is substituted with the valine residue of the 24th glutamine residue, the 30th leucine residue as the valine residue, the 31st alanine residue as the lysine residue and the 35th serine residue as the alanine As the residue, the 37th methionine residue is referred to as a valine residue, the 39th isoleucine residue as a valine residue, the 56th isoleucine residue as a valine residue, the 57th lysine residue as an arginine residue, the 59th threonine residue as alanine The threonine residue at the 95th position is substituted with the threonine residue, the 99th position of the phenylalanine residue is replaced with the tyrosine residue, the 101st position of the glutamine residue is replaced with glutamic acid As the residue, the 106th threonine residue is referred to as an arginine residue, the 107th proline residue as a serine residue, the 108th aspartic acid The humanized R198 heavy chain designed with the substitution of the glutamic acid residue as the glutamic acid residue, the 110th serine residue as the threonine residue, the 130th valine residue as the threonine residue and the 131st methionine residue as the leucine residue was designated " hR198_H1 & Soul ".

The nucleotide sequence coding for the hR198_H1 type heavy chain is SEQ ID NO: 38 in the sequence listing, the nucleotide sequence numbers 58 to 1398 for coding the mature middle sequence deleted from the signal sequence, and the nucleotide numbers 58 to 408 for coding the variable region. The amino acid sequence of the hR198_H1 type heavy chain is SEQ ID NO: 39, the signal sequence is deleted, and the mature heavy chain is composed of the amino acid numbers 20 to 466, and the variable region has the amino acid numbers 20 to 136 thereof. The arrangement of SEQ ID NOs: 38 and 39 is also shown in FIG. 26, respectively. Each CDR array and corresponding array number are shown in FIG.

6) -4-2 hR198_H2 type double jump:

The amino acid of the cR198 heavy chain represented by SEQ ID NO: 29 in the sequence listing is substituted with the valine residue of the 24th glutamine residue, the 30th leucine residue as the valine residue, the 31st alanine residue as the lysine residue and the 35th serine residue as the alanine The arginine residue at the 59th position, the arginine residue at the 59th position, the arginine residue at the 59th position, the arginine residue at the 59th position, the arginine residue at the 59th position, and the arginine residue at the 37th position, As the residue, the 86th lysine residue is referred to as arginine residue, the 95th serine residue as threonine residue, the 99th phenylalanine residue as tyrosine residue, the 101st glutamine residue as glutamic acid residue, the 106th threonine residue as arginine As residue, the 107th proline residue is serine residue, the 108th aspartic acid residue is replaced with glutamic acid residue, the 110th residue The lean residue to threonine residue, a valine residue in the 130th threonine residue, the humanized heavy chain R198 is designed to involve a substitution of a methionine residue to the 131st isoleucine residue was designated as "hR198_H2 type heavy chain".

The nucleotide sequence encoding the hR198_H2 type heavy chain is SEQ ID NO: 40 in the sequence listing, the nucleotide numbers 58 to 1398 encoding the mature heavy chain with the signal sequence excluded, and the nucleotide numbers 58 to 408 encoding the variable region. The amino acid sequence of the hR198_H2 type double-headed horn is SEQ ID NO: 41 in the sequence listing, the mature middle horn head with the signal sequence excluded has the amino acid number 20 to 466, and the variable region has the amino acid number 20 to 136. The arrangement of the array Nos. 40 and 41 is also shown in Fig. 27, respectively. Each CDR array and corresponding array number are shown in FIG.

6) -4-3 hR198_H3 type double jump:

The amino acid of the cR198 heavy chain represented by SEQ ID NO: 29 in the sequence listing is substituted with the valine residue of the 24th glutamine residue, the 30th leucine residue as the valine residue, the 31st alanine residue as the lysine residue and the 35th serine residue as the alanine As the residue, the 37th methionine residue is referred to as a valine residue, the 39th isoleucine residue as a valine residue, the 50th serine residue as an alanine residue, the 56th isoleucine residue as a valine residue, the 57th lysine residue as arginine The threonine residue at position 59 is substituted with alanine residue, the threonine residue at position 60 is substituted with proline residue, the isoleucine residue at position 67 is substituted with valine residue, the 70th valine residue with isoleucine residue, the 81st glutamic acid with alanine residue , The 82th lysine residue as an arginine residue, the 86th lysine residue as an arginine residue, the 95th serine residue as a threonine residue, the 99th The glutamic acid residue at the 101st position is substituted with glutamic acid residue, the 106th threonine residue is substituted with arginine residue, the 107th proline residue with serine residue, the 108th aspartic acid residue with glutamic acid residue, The humanized R198 heavy chain designed with the substitution of the 110th serine residue with a threonine residue, the 130th valine residue with a threonine residue and the 131st methionine residue with a leucine residue was named "hR198_H3 type heavy chain".

The nucleotide sequence encoding the hR198_H3 type heavy chain is SEQ ID NO: 42 in the sequence listing, the nucleotide numbers 58 to 1398 encoding the mature heavy chain in which the signal sequence is excluded, and the nucleotide numbers 58 to 408 encoding the variable region. The amino acid sequence of the hR198_H3 type heavy chain is SEQ ID NO: 43, the signal sequence is deleted, and the mature heavy chain has the amino acid number 20 to 466, and the variable region has the amino acid number 20 to 136. The arrangement of the array Nos. 42 and 43 is also shown in Fig. 28, respectively. Each CDR array and corresponding array number are shown in FIG.

6) -4-4 hR198_H4 type double jump:

The amino acid of the cR198 heavy chain represented by SEQ ID NO: 29 in the sequence listing is substituted with the valine residue of the 24th glutamine residue, the 30th leucine residue as the valine residue, the 31st alanine residue as the lysine residue and the 35th serine residue as the alanine As the residue, the 37th methionine residue is referred to as a valine residue, the 39th isoleucine residue as a valine residue, the 50th serine residue as an alanine residue, the 57th lysine residue as an arginine residue, the 59th threonine residue as alanine As the residue, the threonine residue at the 60th position is substituted with the proline residue, the isoleucine residue at the 67th position is substituted with the valine residue, the 70th valine residue with the isoleucine residue, the 81st glutamic acid with the alanine residue, the 82th lysine residue with the arginine residue , The 86th lysine residue as an arginine residue, the 95th serine residue as a threonine residue, the 99th phenylalanine residue as a tyrosine residue , The 101st glutamine residue as a glutamic acid residue, the 106th threonine residue as an arginine residue, the 107th proline residue as a serine residue, the 108th aspartic acid residue as a glutamic acid residue, the 110th serine residue as a threonine residue , The humanized R198 heavy chain designed with the substitution of the 130th valine residue as a threonine residue and the 131st methionine residue as a leucine residue was named " hR198_H4 type heavy chain. &Quot;

The nucleotide sequence encoding the hR198_H4 type heavy chain is SEQ ID NO: 44 in the sequence listing, the nucleotide numbers 58 to 1398 encoding the mature heavy chain with the signal sequence excluded, and the nucleotide numbers 58 to 408 encoding the variable region. The amino acid sequence of the hR198_H4 type heavy chain is the sequence number 45 of the sequence listing, the mature middle string deleted of the signal sequence has the amino acid number 20 to 466, and the variable region has the amino acid number 20 to 136 thereof. The arrangement of SEQ ID NOS: 44 and 45 is also shown in FIG. 29, respectively. Each CDR array and corresponding array number are shown in FIG.

[Example 7] Production of humanized anti-human Orai1 antibody

7) -1 Construction of humanized anti-human Orai1 antibody light chain hR198_L expression vector

7) Construction of -1-1 hR198_L1 expression vector

A DNA fragment comprising an array coding for a variable region of hR198_L1 as shown in nucleotide numbers 38 to 402 of the nucleotide sequence of hR198_L1 shown in SEQ ID NO: 30 of the sequence listing was synthesized (GENEART Corp. Artificial Gene Synthesis Service). The thus-synthesized DNA fragment was digested with restriction enzymes AvaI and EcoRV, and the hR198_L1 expression vector was constructed by inserting the chimeric and humanized antibody light chain expression vector pCMA-LK into the cleaved site with the same restriction enzymes. The obtained expression vector was named " pCMA-LK / hR198_L1 ".

7) -1-2 Construction of hR198_L2 expression vector

A DNA fragment comprising an array coding for a variable region of L2 represented by nucleotide numbers 38 to 402 of the nucleotide sequence of hR198_L2 shown in SEQ ID NO: 32 in the sequence listing was synthesized (GENEART Corp. Artificial Genome Synthesis Service). Using the synthesized DNA fragment as a template, the DNA fragment containing the sequence encoding the variable region of L2 was amplified with KOD-PLUS- (TOYOBO), digested with restriction enzyme BsiWI, and the chimeric and humanized antibody light chain expression vector pCMA -LK was inserted at the site cleaved with restriction enzyme BsiWI to construct L2 expression vector. The obtained expression vector was named " pCMA-LK / L2 ".

7) Construction of -1-3 hR198_L3 and hR198_L4 expression vectors

A DNA fragment comprising an array encoding the variable region of hR198_L4 shown in nucleotide numbers 38 to 402 of the nucleotide sequence of hR198_L4 shown in SEQ ID NO: 36 of the sequence listing was synthesized (GENEART Corp. Artificial Gene Synthesis Service). HR198_L4 expression vector was constructed by the same method as in Example 7-1-2). The obtained expression vector was named " pCMA-LK / hR198_L4 ".

Using a QuikChange Site-Directed Mutagenesis Kit (Stratagene) with pCMA-G1 / hR198_L4 as a template, a mutation that substitutes a valine residue at amino acid No. 64 with a proline residue was introduced. The resulting expression vector containing the nucleotide sequence shown in SEQ ID NO: 34 in the sequence listing was named " pCMA-G1 / hR198_L3 ".

7) -2 Construction of humanized anti-human Orai1 antibody heavy chain hR198_H expression vector

7) -2-1 Construction of hR198_H1 and hR198_H2 expression vectors

A DNA fragment containing an array encoding the variable region of H0 shown in nucleotide numbers 36 to 425 of the nucleotide sequence of H0 shown in SEQ ID No: 111 of the sequence listing of the candidate humanized candidate for anti-Orai1 antibody heavy chain hR198 was synthesized Gene synthesis service). The DNA fragment containing the sequence encoding the variable region of KOD-plus-H0 was amplified using the synthesized DNA fragment as a template, and the chimeric and humanized antibody IgG1 type heavy chain expression vector pCMA-G1 was digested with restriction enzyme BlpI The H0 expression vector was constructed by inserting one site using an In-Fusion HD PCR Cloning Kit (CLONTECH). The obtained expression vector was named " pCMA-G1 / R198_H0 ". The amino acid sequence of H0 is shown in SEQ ID NO: 112.

Next, a mutation that substitutes the tyrosine residue at the 66th amino acid position of the tryptophan residue was introduced using the KOD-kit-mutagenesis kit (TOYOBO) using pCMA-G1 / R198_H0 as a template. The resulting expression vector containing the nucleotide sequence shown in SEQ ID NO: 40 of the sequence listing was named " pCMA-G1 / hR198_H2 ".

Subsequently, a mutation that substitutes the isoleucine residue at amino acid No. 56 with a valine residue using a QuikChange Site-Directed Mutagenesis Kit (Stratagene) and the following primer set using pCMA-G1 / R198_H2 as a template . The obtained expression vector containing the nucleotide sequence shown by SEQ ID NO: 38 in the sequence listing was named " pCMA-G1 / hR198_H1 ".

7) -2-2 Construction of hR198_H3 and hR198_H4 expression vectors

A DNA fragment containing an array coding for a variable region of H5 represented by nucleotide Nos. 36 to 425 of the nucleotide sequence of H5 shown in SEQ ID NO: 113 of the humanized candidate sequence of anti-Orai1 antibody heavy chain hR198 was synthesized (GENEART Co., Gene synthesis service). H5 expression vector was constructed in the same manner as in Example 7) -2-1. The obtained expression vector was named " pCMA-G1 / hR198_H5 ". The amino acid sequence of H5 is shown in SEQ ID NO: 114.

Subsequently, a mutation for substituting tyrosine residue and valine residue for the 66th amino acid tryptophan residue and the 67th isoleucine residue, respectively, was introduced using pCMA-G1 / H5 as a template using the KOD-Plus-Muta Genesis Kit . The resulting expression vector containing the nucleotide sequence shown in SEQ ID NO: 44 was named "pCMA-G1 / hR198_H4".

Next, using pCMA-G1 / hR198_H4 as a template, a mutation that substitutes the isoleucine residue at amino acid No. 56 in the valine residue was introduced in the same manner as in Example 7) -2-1 and the nucleotide sequence shown in SEQ ID NO: 44 The resulting expression vector containing the sequence was named " pCMA-G1 / hR198_H3 ".

7) -3 Preparation of humanized anti-human Orai1 antibody hR198

4) -5-1, a humanized anti-human Orai1 antibody heavy chain expression vector and a humanized anti-human Orai1 antibody light chain chain expression vector were transfected into FreeStyle 293F cells to obtain a culture supernatant containing the antibody.

The combination of pCMA-G1 / hR198_H1 and pCMA-LK / hR198_L1, pCMA-G1 / hR198_H2 and pCMA-LK / hR198_L2, pCMA-G1 / hR198_H3 and pCMA-LK / hR198_L3, pCMA-G1 / hR198_H4 and pCMA- The humanized anti-human Orai1 antibodies obtained by the above-described methods were named "hR198_H1 / L1", "hR198_H2 / L2", "hR198_H3 / L3" and "hR198_H4 / L4", respectively.

4) The obtained culture supernatant was purified by r-protein A affinity chromatography in the same manner as in -5-2) to obtain a purified antibody sample.

[Example 8] In vitro activity of humanized anti-human Orai1 antibody

8) -1 flow cytometry of humanized anti-human Orai1 antibody

In order to evaluate the binding specificity to human Orai1, the cell suspension of pcDNA3.1-DEST-introduced HEK293T or pcDNA3.1-hOrai1-introduced HEK293T prepared by the method shown in 1) -4-1 was centrifuged and the supernatant was removed And then incubated at 4 ° C for 30 minutes with the addition of the humanized anti-Orai1 antibodies hR198_H1 / L1, hR198_H2 / L2, hR198_H3 / L3, hR198_H4 / L4 and parent moieties cR118 and cR198 Respectively. After washing twice with PBS containing 5% FBS, the anti-human IgG FITC conjugate diluted 100-fold with PBS containing 5% FBS was added, suspended, and allowed to stand at 4 DEG C for 30 minutes. Washed twice with PBS containing 5% FBS, resuspended in PBS containing 5% FBS containing 1 μg / ml propidium iodide, and detection was carried out with a flow cytometer (FC500). Data analysis was performed with Flowjo. After the propidium iodide positive cells were excluded from the gates, a histogram of the FITC fluorescence intensity of the live cells was prepared and the average fluorescence intensity (MFI) was calculated. The humanized anti-human Orai1 antibodies hR198_H1 / L1, hR198_H2 / L2, hR198_H3 / L3 and hR198_H4 / L4 do not bind to pcDNA3.1-DEST-introduced HEK293T cells as in the case of cR118 and cR198 as parent moieties. 1-hOrai1-introduced HEK293T cells were bound in a concentration-dependent manner, and thus they specifically showed binding to human Orai1.

8) -2 Inhibitory effect of humanized anti-human Orai1 antibody on human T cell line activation

Human T cell line, the Jurkat cells, 10% FBS, 100 U / ㎖ penicillin and 100 ㎍ / ㎖ in RPMI1640 containing streptomycin, 1.5 x 10 ⁶ cells / ㎖ was prepared at a concentration of 80 in a 96-well cell culture plate And 10 μl / well of humanized anti-human Orai1 antibodies hH1 / L1, hH2 / L2, hH3 / L3, hH4 / L4 and human chimeric anti-human Orai1 antibodies cR118 and cR198 were added thereto at 37 ° C and 5 % CO _{2 for} 60 min. Thereafter, 10 ng / well of 100 ng / ml PMA and 1 μg / ml of A23187 was added, well stirred, and then cultured at 37 ° C. and 5% CO _{2 for} about 16 hours. After the plate was well stirred, it was centrifuged at 600 g for 3 minutes, and the concentration of IL-2 contained in the supernatant was measured by ELISA. Figure 6 shows that the humanized anti-human Orai1 antibody inhibits the release of IL-2 from Jurkat cells treated with PMA and A23187 in an additive concentration-dependent manner. The humanized anti-human Orai1 antibody inhibited the release of IL-2 from Jurkat cells in an additive concentration-dependent manner, and the inhibitory activity of hH1 / L1 and hH2 / L2 was equivalent to that of human chimeric anti-human Orai1 antibodies cR118 and cR198. On the other hand, hH3 / L3 and hH4 / L4 showed lower inhibitory activity than cR118 and cR198.

[Example 9] Identification of activity-enhancing mutation by ribosome display

9) -1 H chain and L chain libraries

A library in which a mutation was introduced into the H chain or the L chain using the humanized anti-human Orai1 antibody hR198_H4 / L4 as a template was constructed and provided for identification of mutation in activity enhancement by ribosome display.

9) Construction of -1-1 H chain library

80 cycles of PCR were carried out using the following primer set and rTaq DNA polymerase (TOYOBO) with the hR198_H4 gene as a template, and the mutation was randomly introduced into the gene region.

Primer set

5'-ATGCAAGTCCAACTGGTTCAATC-3 '(SEQ ID NO: 46: primer Orai1 HF)

5'-TGACGGAGCCAGCGGGAAGAC-3 '(SEQ ID NO: 47: primer Orai1 CH FR)

Next, the 5 'UTR region containing the H chain gene and the T7 promoter randomly introduced into the mutation, and the TolA gene fragment added with c-Myc at the 5' side and the SecM sequence at the 3 ' Overlap PCR was performed using a primer set to prepare an H chain library.

Primer set

5'-CAGGAAACAGCTATGACCATG-3 '(SEQ ID NO: 48: primer M13 rev long)

5'-CTCGAGTTATTCATTAGGTGAGGCGTTGAGG-3 '(SEQ ID NO: 49: primer SecM Stop R)

9) -1-2 Production of L chain library

80 cycles of PCR were carried out using the following primer set and rTaq DNA polymerase with the hR198_L4 gene as a template, and random mutations were introduced into the gene region.

Primer set

5'-ATGGACATTCAACTGACCCAAAGC-3 '(SEQ ID NO: 50: primer Orai1 Lc F)

5'-GATAAAAACACTCGGGGCCGCCAC-3 '(SEQ ID NO: 51: primer Orai1 CL-FR)

9) An overlapping PCR was carried out using the L chain gene into which the mutation was introduced at random and the two gene fragments as templates as in the description of -1-1, thereby preparing an L chain library.

9) Production of -1-3 H chain gene fragment

Using the hR198_H4 gene as a template, PCR was performed using the following primer set and KOD-plus- to amplify the H chain gene region.

Primer set

5'-ATGCAAGTCCAACTGGTTCAATC-3 '(SEQ ID NO: 46: primer Orai1 HF)

5'-TCATTATTTGTCATCGTCATCTTTATAGTCGAATTCTTCGCCACGATTAAAGGATTTGGTGAC-3 '(SEQ ID NO: 52: primer Orai1 HR-FLAG R)

Next, overlap PCR was carried out using the following primer sets using the 5'UTR region containing the gene fragment and the T7 promoter as templates, to prepare an H chain gene fragment.

Primer set

5'-CAGGAAACAGCTATGACCATG-3 '(SEQ ID NO: 48: primer M13 rev long)

5'-TCATTATTTGTCATCGTCATCTTTATAGTCGAATTCTTCGCCACGATTAAAGGATTTGGTGAC-3 '(SEQ ID NO: 52: primer Orai1 HR-FLAG R)

9) Production of -1-4 L chain gene fragment

Using the hR198_L4 gene as a template, PCR was carried out using the following primer set and KOD-plus- to amplify the L chain gene region.

Primer set

5'-ATGGACATTCAACTGACCCAAAGC-3 '(SEQ ID NO: 50: primer Orai1 Lc F)

5'-TCATTATTTGTCATCGTCATCTTTATAGTCGAATTCTTCGCCACGATTAAAGGATTTGGTGAC-3 '(SEQ ID NO: 53: primer Orai1 CL-FLAG R)

Next, overlap PCR was performed in the same manner as described in 9) -1-1 to prepare an L chain gene fragment.

Primer set

5'-CAGGAAACAGCTATGACCATG-3 '(SEQ ID NO: 48: primer M13 rev long)

5'-TCATTATTTGTCATCGTCATCTTTATAGTCGAATTCTTCGCCACGATTAAAGGATTTGGTGAC-3 '(SEQ ID NO: 53: primer Orai1 CL-FLAG R)

9) -1-5 Preparation of mRNA

Each mRNA was synthesized with the T7 RiboMax Express Large Scale RNA Production System (Promega) using the library and gene fragment prepared in Example 9-1-1 to 9-1-4 as a template.

9) -2 Screening with ribosome display

The H chain library and the L chain gene fragment were combined to form an H chain ribosome display Fab, and the L chain library and the H chain gene fragment were combined to prepare an L chain ribosome display Fab. 20 pmol of H chain (or L chain) mRNA and 100 pmol of ribosome were added to the PUREfrex reaction solution (GeneFrontier), and the mixture was kept at 30 ° C for 45 minutes. Similarly, 40 pmol of L chain (or H chain) mRNA and 200 pmol of ribosome were added to the PUREfrex reaction solution and incubated at 30 ° C for 45 minutes. Next, a H chain (or L chain) ribosomal display Fab was prepared by mixing the reaction solution after translation of the H chain (or L chain) and the L chain (or H chain) and further incubating at 30 ° C for 90 minutes , And then the reaction was stopped by cooling to 4 占 폚. Subsequently, the antigen was added to the reaction solution, and the mixture was agitated at 4 DEG C for 1 hour to bind Fab and the antigen. Examples of the antigen include (1) a sample in which a formalin-immobilized human Orai1-expressing CHO cell established using pcDNA3.1-hOrai1 prepared in (1-1) -1-2 or a biotin-PEGylated human Orai1 loop region peptide (SIGMA) was used.

Biotin PEGylated human Orai1 loop region peptide

Biotin-PEG-SGSGFLPLKKQPGQPRPTSKPPASGAAANVSTSGITPGQAAAIASTTI (SEQ ID NO: 115)

Antibody-bound ribosomal display Fabs were recovered with Nonolink Streptavidin magnetic beads (SoluLink). Subsequently, the antigen was suspended in 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 15 mM Mg (OAc) ₂ , 0.05% Tween 20, 1 mg / ml yeast RNA and 50 mM Tris- , 15 mM Mg (OAc) ₂ , and 0.05% Tween20. Then, 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 15 mM Mg (OAc) ₂ and 50 mM EDTA were added to the antigen, and the mixture was allowed to stand at room temperature for 10 minutes and then the supernatant containing mRNA was recovered by centrifugation . The recovered mRNA was transformed into cDNA by Transcriptor High Fidelity cDNA Synthesis Kit (Roche), and then PCR was carried out using the following primer set and KOD-Plus - to amplify it as DNA. MRNA was synthesized using the DNA as a template and subjected to the same screening. By performing this screening cycle a plurality of times, an antibody gene that binds strongly to the antigen was selected.

Primer set

5'-ATGGACATTCAACTGACCCAAAGC-3 '(SEQ ID NO: 50: primer Orai1-LcF)

5'-CAGATCCTCCTCAGAGATCAGCTTCTGCTC-3 '(SEQ ID NO: 54: primer Myc-R)

9) -3 Preparation of Fab Protein

The selected gene was subcloned into a vector for E. coli expression, and a clone with improved binding to human Orai1 always expressing CHO cells was screened by Cell ELISA. First, the DNA after selection was digested with restriction enzymes EcoRV and XhoI, inserted into a Fab expression vector (GeneFrontier) digested with the same enzyme, and then introduced into Escherichia coli BL21 (DE3). The transformants were cultured on a round bottom 96 well plate at 37 째 C for 4-5 hours with 150 ㎕ carbenicillin / 0.1% glucose / 2 x YT per well. Next, the plate was cooled to 4 캜, IPTG was added to give a final concentration of 0.5 mM, and the plate was incubated with shaking at 30 캜 for one night. Next, the cells were recovered by centrifugation, and then lysis buffer (2.5 mg / ml Lysotyme, 100 U DNase I) was added and the mixture was shaken at room temperature for 60 minutes. Subsequently, the supernatant was recovered by centrifugation to obtain a Fab sample.

9) Screening by -4 Cell ELISA

After incubation with the human full-sheet Orai1 expressing cells in the plate 384, the cleaning buffer, and washed with (PBS (), 20 mM MgSO 4, 2.5% FBS). Next, a Fab sample was added to the plate, and the plate was shaken at 4 DEG C for 1 hour. After washing four times with a washing buffer, peroxidase-labeled anti-human F (ab ') ₂ goat antibody (Jackson Immuno Research Co.) was added and the mixture was shaken at 4 ° C for 30 minutes. After washing three times with washing buffer, it was washed three times with PBS (-) and 20 mM MgSO ₄ . Then, a coloring reagent (0.4 mg / ml Tetramethyl-benzimine, 200 mM sodium acetate (pH 3.4), 0.01% hydrogen peroxide water) was added and the mixture was shaken at room temperature for 15 minutes. After addition of 2 N HCl, OD ₄₅₀ was measured. Fab samples that showed higher OD ₄₅₀ than human Orai1 always expressing CHO cells were selected compared to control CHO cells.

9) -5 Antigen binding activity of anti-human Orai1 antibody Fab by flow cytometry

In order to evaluate the binding specificity to human Orai1, the cell suspension of pcDNA3.1-DEST-introduced HEK293T or pcDNA3.1-hOrai1-introduced HEK293T prepared by the method shown in 1) -4-1 was centrifuged and the supernatant was removed After incubation, the light chain variable clones LCDR60, LCDR67, LCDR83, CE151, PE057 and heavy chain mutation clones HCDR046, HCDR047, HEP087, HEP124, HEP237 and hR198_H4 / L4-Fab And the mixture was allowed to stand at 4 占 폚 for 30 minutes. After washing twice with PBS containing 5% FBS, an anti-human IgG FITC conjugate diluted 100-fold with PBS containing 5% FBS was added, suspended, and allowed to stand at 4 DEG C for 30 minutes. Washed twice with PBS containing 5% FBS, resuspended in PBS containing 5% FBS containing 1 μg / ml propidium iodide, and detection was carried out with a flow cytometer (FC500). Data analysis was performed with Flowjo. After removing the propidium iodide positive cells, the histogram of the FITC fluorescence intensity of viable cells was prepared and the average fluorescence intensity (MFI) was calculated. HCD194, HEP147, HEP124, and HEP237, as well as the hR198_H4 / L4-Fab of the parent antibody Fab, bind to the pcDNA3.1-DEST-introduced HEK293T cells As shown in Fig. 7, the HEK293T cells transfected with pcDNA3.1-hOrai1 showed a tendency to bind to human Orai1 in equal or greater amount as compared with the parent antibody Fab.

[Example 10] Production of affinity-matched antibody of humanized anti-human Orai1 antibody

Several mutations that enhance activity identified in the light chain variable clones LCDR60, LCDR67, LCDR83, CE151, PE057 and heavy chain mutation clones HCDR046, HCDR047, HEP087, HEP124 and HEP237 selected by Example 9 were designated as hR198_H3 / L3 , More than 100 kinds of modified hR198_H3 / L3 antibodies were prepared and evaluated in terms of binding affinity, in vitro activity, productivity, and heterologous antigenicity to humans. As a result, the following antibodies were selected.

10) -1 Design of affinity-matched antibody of humanized anti-human Orai1 antibody

10) -1-1 hR198_LG1 type light chain:

A humanized R198 designed with the substitution of an asparagine residue at the 51st amino acid position in the hR198_L3 light chain shown in SEQ ID NO: 35 of the sequence listing with a glycine residue, a 113th threonine residue as an isoleucine residue, and a 117th threonine residue as a serine residue The light chain was named " hR198_LG1 type light chain ".

The nucleotide sequence encoding the hR198_LG1 type light chain is SEQ ID NO: 55 in the sequence listing, the nucleotide sequence numbers 61 to 702 encoding the mature light chain in which the signal sequence is excluded, and the nucleotide numbers 61 to 378 encoding the variable region. The amino acid sequence of the hR198_LG1 type light chain is SEQ ID NO: 56 in the sequence listing, the mature light chain in which the signal sequence is excluded is in the amino acid number 21 to 234, and the variable region is in the amino acid number 21 to 126 in the amino acid sequence. The arrangement of SEQ ID NOS: 55 and 56 is also shown in FIG. 30, respectively. Each CDR array and corresponding array number are shown in FIG.

10) -1-2 hR198_LG2 type light chain:

The arginine residue at position 44 of the hR198_L3 light chain shown in SEQ ID NO: 35 of the sequence listing is substituted with a histidine residue, the 48th serine residue with an asparagine residue, the 51st asparagine residue with a glycine residue, Which is designed to carry out the substitution of the 75th glutamic acid residue as an aspartic acid residue, the 76th serine residue as a tryptophan residue, the 113th threonine residue as an isoleucine residue, and the 117th threonine residue as a serine residue, The light chain of R198 was named " hR198_LG2 type light chain ".

The nucleotide sequence encoding the hR198_LG2 type light chain is SEQ ID NO: 57 in the sequence listing, the nucleotide sequence numbers 61 to 702 encoding the mature light chain in which the signal sequence is excluded, and the nucleotide numbers 61 to 378 encoding the variable region. The amino acid sequence of the hR198_LG2 type light chain is SEQ ID NO: 58 in the sequence listing, the mature light chain in which the signal sequence is excluded consists of the amino acid numbers 21 to 234, and the variable region has the amino acid numbers 21 to 126 thereof. The arrangement of the array Nos. 57 and 58 is also shown in Fig. 31, respectively. Each CDR array and corresponding array number are shown in FIG.

10) -1-3 hR198_LG3 type light chain:

The arginine residue at position 44 in the hR198_L3 light chain shown in SEQ ID NO: 35 of the sequence listing is substituted with a histidine residue, the 47th glutamine residue with arginine residue, the 48th serine residue with asparagine residue, the 51st asparagine residue with glycine The seventh serine residue is referred to as a leucine residue, the 73th threonine residue as a serine residue, the 75th glutamic acid residue as an aspartic acid residue, the 76th serine residue as a tryptophan residue, and the 113th threonine residue as a The humanized R198 light chain designed with the isoleucine residue replaced with the serine residue at the 117th threonine residue was named " hR198_LG3 type light chain ".

The nucleotide sequence encoding the hR198_LG3 type light chain is SEQ ID NO: 59 in the sequence listing, the nucleotide sequence numbers 61 to 702 encoding the mature light chain in which the signal sequence is excluded, and the nucleotide numbers 61 to 378 encoding the variable region. The amino acid sequence of the hR198_LG3 type light chain is SEQ ID NO: 60, the signal sequence is mature light chain having the amino acid number 21 to 234, and the variable region is the amino acid number 21 to 126. The arrangement of SEQ ID NOS: 59 and 60 is also shown in Fig. 32, respectively. Each CDR array and corresponding array number are shown in FIG.

10) -1-4 hR198_HG1 type double jump:

The humanized R198 heavy chain designed with the substitution of the aspartic acid residue at position 78 of the amino acid number 78 in the heavy chain of hR198_H3 in SEQ ID NO: 43 and the alanine residue at position 123 with the aspartic acid residue was designated as "hR198_HG1 type light chain" .

The nucleotide sequence encoding the hR198_HG1 type heavy chain is SEQ ID NO: 61 in the sequence listing, the nucleotide numbers 58 to 1398 encoding the mature heavy chain with the signal sequence excluded, and the nucleotide numbers 58 to 408 encoding the variable region. The amino acid sequence of the hR198_HG1 type heavy chain is represented by SEQ ID NO: 62 in the sequence listing, the mature heavy chain having the signal sequence excluded is composed of amino acid numbers 20 to 466, and the variable region is composed of amino acid numbers 20 to 135 thereof. The arrangement of the array Nos. 61 and 62 is also shown in Fig. 33, respectively. Each CDR array and corresponding array number are shown in FIG.

10) -1-5 hR198_HG2 type double jump:

The amino acid residue at position 48 of the hR198_H3 heavy chain shown in SEQ ID NO: 43 in the sequence listing is substituted with isoleucine residue, the 78th asparagine residue is replaced with an aspartic acid residue, the 81st alanine residue with a glycine residue, The humanized R198 heavy chain designed with substitution with an alanine residue was named " hR198_HG2 type light chain ".

The nucleotide sequence encoding the hR198_HG2 type heavy chain is SEQ ID NO: 63 in the sequence listing, the nucleotide sequence numbers 58 to 1398 encoding the mature heavy chain having the signal sequence excluded, and the nucleotide numbers 58 to 408 encoding the variable region. The amino acid sequence of the hR198_HG2 type double-headed horn is the sequence number 64 in the sequence listing, the mature middle horn head with the signal sequence excluded has the amino acid number 20 to 466, and the variable region has the amino acid number 20 to 135. The arrangement of array Nos. 63 and 64 is also shown in Fig. 34, respectively. Each CDR array and corresponding array number are shown in FIG.

10) -1-6 hR198_HG3 type double jump:

The amino acid residue at position 48 of the hR198_H3 heavy chain shown in SEQ ID NO: 43 in the sequence listing is substituted with an isoleucine residue, the 78th asparagine residue is replaced with an aspartic acid residue, the 81st alanine residue with a methionine residue, The humanized R198 heavy chain designed with substitution with an alanine residue was named " hR198_HG3 type light chain ".

The nucleotide sequence encoding the hR198_HG3 type heavy chain is SEQ ID NO: 65 in the sequence listing, the nucleotide sequence numbers 58 to 1398 encoding the mature heavy chain with the signal sequence excluded, and the nucleotide numbers 58 to 408 encoding the variable region. The amino acid sequence of the hR198_HG3 type heavy chain is SEQ ID NO: 66, the signal sequence is deleted, and the mature heavy chain is composed of the amino acid numbers 20 to 466, and the variable region has the amino acid numbers 20 to 135 thereof. The arrangement of SEQ ID NOS: 65 and 66 is also shown in FIG. 35, respectively. Each CDR array and corresponding array number are shown in FIG.

10) -2 Production of an affinity-matched antibody expression vector for humanized anti-human Orai1 antibody

10) -2-1 Construction of hR198_LG1 type light chain expression vector

Using the pCMA-LK / hR198_L3 as a template, the asparagine residue at amino acid number 51 was substituted with glycine residue, the threonine residue at residue 113 was substituted with isoleucine residue, the 117 th threonine residue was replaced with serine residue . ≪ / RTI > The resulting expression vector containing the nucleotide sequence shown in SEQ ID NO: 55 of the sequence listing was named " pCMA-LK / hR198_LG1 ".

10) -2-2 Construction of hR198_LG2 and LG3 type light chain expression vectors

A DNA fragment comprising an array coding for a variable region of hR198_LG3 shown in nucleotide numbers 38 to 402 of the nucleotide sequence of LG3 shown in SEQ ID NO: 59 of the sequence listing was synthesized (GENEART Corp. Artificial Gene Synthesis Service). The synthesized DNA fragment was digested with restriction enzymes EcoRV and AvaI and inserted into pCMA-LK / hR198_LG1 digested with the same restriction enzymes to construct hR198_LG3 expression vector. The obtained expression vector was named " pCMA-LK / hR198_LG3 ".

A mutation that substitutes the arginine residue of amino acid number 47 for the glutamine residue and the 73 residue of the serine residue for the threonine residue was introduced by using pCMA-LK / hR198_LG3 as a template using KOD-plus-mutagenesis. The resulting expression vector containing the nucleotide sequence shown in SEQ ID NO: 57 of the sequence listing was named " pCMA-LK / hR198_LG2 ".

10) -2-3 Construction of hR198_HG1 type heavy chain expression vector

A mutation that substitutes the aspartic acid residue at amino acid number 78 for the aspartic acid residue and the 123th valine residue at the alanine residue was introduced using pCMA-G1 / hR198_H3 as a template using KOD-plus-mutagenesis. The resulting expression vector containing the nucleotide sequence shown in SEQ ID NO: 61 of the sequence listing was named " pCMA-G1 / hR198_HG1 ".

10) -2-4 Construction of hR198_HG2 type heavy chain expression vector

Using mutation of pCMA-G1 / hR198_HG1 as a template, a mutation in which a valine residue at amino acid number 48 was substituted with an isoleucine residue and an 81-th alanine residue was replaced with a glycine residue was introduced using KOD-plus-mutagenesis. The resulting expression vector containing the nucleotide sequence shown in SEQ ID NO: 63 of the sequence listing was named " pCMA-G1 / hR198_HG2 ".

10) -2-5 Construction of hR198_HG3 type heavy chain expression vector

Using a template of pCMA-G1 / hR198_HG2, a mutation for substituting the methionine residue for the glycine residue at amino acid No. 81 was introduced using KOD-plus-mutagenesis. The resulting expression vector containing the nucleotide sequence shown in SEQ ID NO: 65 of the sequence listing was named " pCMA-G1 / hR198_HG3 ".

10) -3 Preparation of affinity-matched antibody of humanized anti-human Orai1 antibody

4) -5-1, the humanized anti-human Orai1 antibody heavy chain expression vector and the humanized anti-human Orai1 antibody light chain chain expression vector prepared in 10) -2 were transfected into FreeStyle 293F cells, Was obtained.

PCMA-G1 / hR198_HG3, pCMA-LK / hR198_LG1, pCMA-LK / hR198_LG2, pCMA-LK / hR198_LG3 and pCMA-G1 / Humanized anti-human Orai1 antibody obtained by the combination was named "hR198_H3 / LG1", "hR198_HG1 / LG1", "hR198_HG1 / LG2", "hR198_HG1 / LG3", "hR198_HG2 / LG1" and "hR198_HG3 / LG1" Respectively.

4) The obtained culture supernatant was purified by r-protein A affinity chromatography in the same manner as in -5-2) to obtain a purified antibody sample.

[Example 11] In vitro activity of an affinity-matched antibody

11) -1 Evaluation of Binding Ability of Affinity Mating Antibody by Flow Cytometry

In order to evaluate the binding specificity to human Orai1, the cell suspension of pcDNA3.1-DEST-introduced HEK293T or pcDNA3.1-hOrai1-introduced HEK293T prepared by the method shown in 1) -4-1 was centrifuged and the supernatant was removed HR198_HG1 / LG1, hR198_HG1 / LG2, hR198_HG1 / LG3, hR198_HG2 / LG1 and hR198_HG3 / LG1 of the affinity-matched antibody produced in 10) -3 and hR198_H3 / L3 and hR198_H4 / L4, and the mixture was allowed to stand at 4 占 폚 for 30 minutes. After washing twice with PBS containing 5% FBS, the anti-human IgG FITC conjugate diluted 100-fold with PBS containing 5% FBS was added, suspended, and allowed to stand at 4 DEG C for 30 minutes. Washed twice with PBS containing 5% FBS, resuspended in PBS containing 5% FBS containing 1 μg / ml propidium iodide, and detection was carried out with a flow cytometer (FC500). Data analysis was performed with Flowjo. After the propidium iodide positive cells were excluded from the gates, a histogram of the FITC fluorescence intensity of the live cells was prepared and the average fluorescence intensity (MFI) was calculated. Similarly to the parental sequences hR198_H3 / L3 and hR198_H4 / L4, pcDNA3.1-DEST-introduced HEK293T cells, hR198_HG1 / LG1, hR198_HG1 / LG1, hR198_HG1 / LG2, hR198_HG1 / LG3, hR198_HG1 / LG3, hR198_HG2 / LG1 and hR198_HG3 / , And as shown in Fig. 8, the HEK293T cells transfected with pcDNA3.1-hOrai1 showed a tendency to bind to human Orai1 in equal or greater amount as compared with the parent antibody.

11) -2 Affinity-matched anti-T-cell activation of the antibody

Human T cell line, the Jurkat cells, 10% FBS, 100 U / ㎖ penicillin and 100 ㎍ / ㎖ in RPMI1640 containing streptomycin, 1.5 x 10 ⁶ cells / ㎖ was prepared at a concentration of 80 in a 96-well cell culture plate HL198_HG1 / LG1, hR198_HG1 / LG2, hR198_HG1 / LG3, hR198_HG2 / LG1, hR198_HG3 / LG1 and humanized anti human Orai1 antibodies hR198_H3 / L3 and hR198_H4 / L4 were cultured in 10 Mu] l / well, and the cells were pretreated at 37 [deg.] C and 5% CO _{2 for} 60 minutes. Thereafter, 10 ng / well of 100 ng / ml PMA and 1 μg / ml of A23187 was added, well stirred, and then cultured at 37 ° C. and 5% CO _{2 for} about 16 hours. After the plate was well stirred, it was centrifuged at 600 g for 3 minutes, and the concentration of IL-2 contained in the supernatant was measured by ELISA. Figure 9 shows that the affinity-matched antibody inhibits the release of IL-2 from Jurkat cells treated with PMA and A23187 in an additive concentration-dependent manner. The affinity-matched antibody inhibited the release of IL-2 from Jurkat cells in an additive concentration-dependent manner, and all of the inhibitory activities exceeded that of the humanized anti-human Orai1 antibodies hH3 / L3 and hH4 / L4.

[Example 12] Fabrication of an effector activity-reducing variant of an affinity-matched antibody

A normal region modified hR198_HG1-LALA sequence substituted for two amino acid residues in the normal region of hR198_HG1 was constructed as described in the following examples.

12) -1 Design of LALA type heavy chain expression vector

In order to avoid cytotoxicity against normal human Orai1 expressing cells, the effector activity of the antibody is preferably low. The effector activity is known to vary depending on the subclass of the antibody. IgG4 has low ADCC and CDC activity, and IgG2 has CDC activity but low ADCC activity. From this characteristic, it is possible to prepare an IgG1 antibody in which ADCC and CDC activity are reduced by replacing the arrangement of a part of the normal region of IgG1 with reference to IgG2 and IgG4. As an example, Marjan Hezareh et. al. According to Journal of Virology, 75 (24): 12161-12168 (2001), when the 234th and 235th leucine residues of IgG1 (EU index by Kabat et al.) Were substituted with alanine residues, ADCC and CDC activity . Thus, for the hR198_HG1 type heavy chain produced in (10) -1, the humanized anti-human Orai1 antibody heavy chain designed with the replacement of the 253th leucine residue with the alanine residue and the 254th leucine residue with the alanine residue Was named " hR198_HG1-LALA type heavy chain ".

12) -2 Construction of LALA type heavy chain expression vector

12) -2-1 Construction of hR198_HG1-LALA type heavy chain expression vector

The hR198_H4-LALA type heavy chain expression vector was constructed by introducing the mutation using the KOD-plus-mutagenesis kit, using the hR198_H4 type heavy chain pCMA-G1 / hR198_H4 template prepared in Example 7-2). The obtained expression vector was named " pCMA-G1-LALA / hR198_H4 ". The nucleotide sequence encoding the hR198_H4-LALA type heavy chain and the amino acid sequence of the heavy chain are shown in SEQ ID NOS: 67 and 68 (FIG. 36), respectively.

An antibody variable region obtained by digesting pCMA-G1 / hR198_HG1 prepared in 10) -2 with the same restriction enzyme was included in a DNA fragment of about 4.2 kb obtained by digesting pCMA-G1-LALA / hR198_H4 with restriction enzymes PstI and XbaI The hR198_HG1-LALA-type double-stranded expression vector was constructed by inserting a DNA fragment of about 0.6 kb in length using Ligation High ver. The obtained expression vector was named " pCMA-G1-LALA / hR198_HG1 ". The nucleotide sequence encoding the hR198_HG1-LALA type heavy chain and the amino acid sequence of the heavy chain are shown in SEQ ID NOS: 69 and 70 (FIG. 37), respectively.

12) -3 Preparation of Affective Activity Reduced Variants of Affinity-Mating Antibodies

12) -3-1 Production of effector activity-reducing variants of affinity-matched antibodies

FreeStyle 293F cells (Life Technologies) were subcultured and cultured according to the manual. 1.2 × 10 ⁹ FreeStyle 293F cells (Life Technologies) from the logarithmic growth phase were inoculated into 3 L Fernbach Erlenmeyer Flask (CORNING), diluted with FreeStyle 293 expression medium (Invitrogen), and adjusted to 1.0 × 10 ⁶ cells / Incubation was carried out at 37 캜 in an 8% CO ₂ incubator at 90 rpm for 1 hour with shaking. 3.6 mg of polyethylenimine (Polyscience # 24765) was dissolved in 20 ml of Opti-Pro SFM (Life Technologies), and then a light chain expression vector (0.8 ㎎) prepared using a PureLink HiPure Plasmid kit (Life Technologies) ) And a heavy chain expression vector (0.4 mg) were added to 20 ml of Opti-Pro SFM (Life Technologies). To 20 ml of a mixture of polyethyleneimine / Opti-Pro SFM, 20 ml of the expression vector / Opti-Pro SFM mixture was added, carefully stirred, and allowed to stand for another 5 minutes before adding to FreeStyle 293F cells. The culture supernatant obtained by shaking culture at 90 rpm for 7 days in an 8% CO ₂ incubator at 37 ° C was filtered with a Disposable Capsule Filter (ADVANTEC # CCS-045-E1H). hR198_HG1 / LG1 was produced by the combination of pCMA-G1 / hR198_HG1 and pCMA-LK / hR198_LG1, and hR198_HG1-LALA / LG1 was produced by the combination of pCMA-G1-LALA / hR198_HG1 and pCMA-LK / hR198_LG1.

12) -3-2 Effectiveness of the affinity-matched antibody Anti-

Example 12) The antibody was purified from the culture supernatant obtained in -3-1 by a two-step process of r-protein A affinity chromatography (under 4 - 6 캜) and ceramic hydroxyapertite (under room temperature). The buffer replacement step after r protein A affinity chromatography purification and after the ceramic hydroxyapatite purification was carried out at 4 - 6 캜. The culture supernatant was applied to MabSelectSuRe (GE Healthcare Bioscience, HiTrap column) equilibrated with PBS. After the culture supernatant was put in the column, the column was washed with PBS having a column capacity of 2 times or more. Next, the fractions containing the antibody were collected by elution with 2 M arginine hydrochloride solution (pH 4.0). The fraction was replaced with PBS by dialysis (Thermo Scientific, Slide-A-Lyzer Dialysis Cassette) and the antibody solution diluted 5-fold with 5 mM sodium phosphate / 50 mM MES / pH 7.0 buffer was washed with 5 mM NaPi (Bio-Scale CHT Type-1 Hydroxyapatite Column) equilibrated with a buffer of 50 mM MES / 30 mM NaCl / pH 7.0. A linear concentration gradient elution with sodium chloride was performed to collect fractions containing the antibody. The fractions were subjected to liquid displacement to HBSor (25 mM histidine / 5% sorbitol, pH 6.0) by dialysis (Thermo Scientific, Slide-A-Lyzer Dialysis Cassette). Concentrated by Centrifugal UF Filter Device VIVASPIN20 (cut-off molecular weight UF10K, Sartorius Inc., 4 ° C), and the IgG concentration was adjusted to 5 mg / ml or more. Finally, the sample was filtered with a Minisart-Plus filter (Sartorius) to obtain a purified sample.

[Example 13] Construction of expression vectors for human anti-human Orai1 antibodies 2C1.1 and 5H3.1

The 2C1.1 antibody and the 5H3.1 antibody were prepared based on the amino acid sequence of light chain and heavy chain described in WO2011063277A1.

13) -1 Construction of chimeric and humanized IgG2 type heavy chain expression vector pCMA-G2

a DNA fragment obtained by digesting pCMA-LK with XbaI and PmeI to remove the κ chain secretion signal and the human κ chain normal region and the DNA fragment encoding the amino acid sequence of human heavy chain secretion signal and human IgG2 normal region shown in SEQ ID NO: Were combined using an In-Fusion Advantage PCR cloning kit to generate a chimeric and humanized IgG2 type heavy chain expression vector pCMA-G2 having a signal sequence, a cloning site, and a human IgG2 heavy chain downstream region downstream of the CMV promoter Respectively.

13) -2 2C1.1 Construction of antibody heavy chain expression vector

A DNA fragment containing an array encoding the variable region of the 2C1.1 antibody heavy chain shown in nucleotide Nos. 36 to 434 of the nucleotide sequence encoding the 2C1.1 antibody heavy chain shown in SEQ ID NO: 72 of the sequence listing was synthesized Artificial gene synthesis service). The DNA fragment containing the sequence encoding the variable region of the KOD-plus-2C1.1 antibody heavy chain was amplified using the synthesized DNA fragment as a template, and the chimeric and humanized antibody IgG2 type heavy chain expression vector pCMA-G2 The 2C1.1 antibody heavy-chain expression vector was constructed by inserting the fragment with the restriction enzyme BlpI using an In-Fusion HD PCR cloning kit. The obtained expression vector was named " pCMA-G2 / 2C1.1 ".

The amino acid sequence of 2C1.1 antibody heavy chain is shown in SEQ ID NO: 73 of the sequence listing.

13) -3 2C1.1 Construction of antibody light chain expression vector

A DNA comprising an array encoding a variable region of the 2C1.1 antibody light chain and a normal region (? Chain) represented by the nucleotide numbers 38 to 739 of the nucleotide sequence encoding the 2C1.1 antibody light chain shown in SEQ ID NO: Fragments were synthesized (GENEART artificial gene synthesis service). The DNA fragment containing the DNA fragment containing the variable region and the normal region of the 2C1.1 antibody light chain of KOD-plus-L as a template was amplified, and the chimeric and humanized light chain variable chain expression vector pCMA-LK Were inserted into restriction enzyme BsiWI and PmeI sites using an In-fusion HD PCR cloning kit to construct a 2C1.1 antibody light chain expression vector. The obtained expression vector was named " pCMA-L / 2C1.1 ".

The amino acid sequence of the 2C1.1 antibody light chain is shown in SEQ ID NO: 75 of the sequence listing.

13) -4 5H3.1 Construction of antibody heavy-chain expression vector

A DNA fragment comprising an array encoding the variable region of the 5H3.1 antibody heavy chain shown in nucleotide Nos. 36 to 434 of the nucleotide sequence encoding the 5H3.1 antibody heavy chain represented by SEQ ID NO: 76 of the sequence listing was synthesized Artificial gene synthesis service). The 5H3.1 antibody heavy chain expression vector was constructed in the same manner as in Example 13) -2. The obtained expression vector was named " pCMA-G2 / 5H3.1 ".

The amino acid sequence of the 5H3.1 antibody heavy chain is shown in SEQ ID NO: 77 of the sequence listing.

13) -5 5H3.1 Construction of antibody light chain expression vector

A DNA fragment containing a variable region of the 5H3.1 antibody light chain shown in the nucleotide numbers 38 to 742 of the nucleotide sequence encoding the 5H3.1 antibody light chain shown in SEQ ID NO: 78 of the sequence listing and a sequence encoding the normal region was synthesized (GENEART Artificial Gene Synthesis Service). 5H3.1 antibody light chain expression vector was constructed in the same manner as in Example 13) -3. The resulting expression vector was named " pCMA-L / 5H3.1 ".

The amino acid sequence of the 5H3.1 antibody light chain is shown in SEQ ID NO: 79 of the sequence listing.

13) -6 Preparation of 2C1.1 antibody and 5H3.1 antibody

13) -6-1 Production of 2C1.1 antibody and 5H3.1 antibody

Example 12) An antibody was produced in the same manner as in -3-1. The 2C1.1 antibody is produced by the combination of pCMA-G2 / 2C1.1 and pCMA-L / 2C1.1, and the 5H3.1 antibody is produced by the combination of pCMA-G2 / 5H3.1 and pCMA-L / 5H3.1 Lt; / RTI >

13) -6-2 2-step purification of 2C1.1 antibody and 5H3.1 antibody

In the same manner as in Example 12) -3-2, the culture supernatant produced in Example 13) -6-1 was subjected to two-step purification of the antibody.

Experimental Example 14 Production of mouse anti-human Orai1 antibodies 10F8, 14F74, and 17F6

10F8 antibody, 14F74 antibody, and 17F6 antibody were prepared based on the amino acid sequence of light chain and heavy chain described in WO2013091903A1.

14) -1 10F8 antibody heavy-chain expression vector

A DNA fragment containing a nucleotide sequence encoding the 10F8 antibody heavy chain represented by SEQ ID NO: 80 of the sequence listing was synthesized (GENEART Corp. Artificial Gene Synthesis Service). A DNA fragment containing the sequence encoding the KOD-plus-10F8 antibody heavy chain was amplified using the synthesized DNA fragment as a template, and the chimeric and humanized antibody light chain expression vector pCMA-LK was digested with restriction enzymes XbaI and PmeI To construct a 10F8 antibody heavy-chain expression vector by inserting the κ chain secretion signal and the human κ chain normal region at the site of cleavage using an In-Fusion HD PCR cloning kit. The obtained expression vector was named " pCMA / 10F8H ".

The amino acid sequence of the 10F8 antibody heavy chain was shown in SEQ ID NO: 81 of the sequence listing.

14) -2 Construction of 10F8 antibody light chain expression vector

A DNA fragment containing a nucleotide sequence encoding the 10F8 antibody light chain shown in SEQ ID NO: 82 of the sequence listing was synthesized (GENEART Strings DNA Fragments). The synthesized DNA fragment was digested with restriction enzymes XbaI and PmeI and the chimeric and humanized antibody light chain expression vector pCMA-LK was digested with restriction enzymes XbaI and PmeI, and the In-Fusion HD PCR cloning kit was used to remove the κ chain secretion signal and the human κ chain normal region To construct a 10F8 antibody light chain expression vector. The obtained expression vector was named " pCMA / 10F8L ".

The amino acid sequence of the 10F8 antibody light chain is shown in SEQ ID NO: 83 of the sequence listing.

14) -3 Construction of 14F74 antibody heavy chain expression vector

A DNA fragment containing a nucleotide sequence encoding the 14F74 antibody heavy chain shown in SEQ ID NO: 84 of the sequence listing was synthesized (GENEART artificial gene synthesis service). The 14F74 antibody heavy chain expression vector was constructed in the same manner as in Example 14) -1. The obtained expression vector was named " pCMA / 14F74H ".

The amino acid sequence of the 14F74 antibody heavy chain is shown in SEQ ID NO: 85 of the sequence listing.

14) -4 Construction of 14F74 antibody light chain expression vector

A DNA fragment containing a nucleotide sequence encoding the 14F74 antibody light chain shown in SEQ ID NO: 86 of the sequence listing was synthesized (GENEART Strings DNA Fragments). The 14F74 antibody light chain expression vector was constructed in the same manner as in Example 14) -2. The obtained expression vector was named " pCMA / 14F74L ".

The amino acid sequence of the 14F74 antibody light chain is shown in SEQ ID NO: 87 of the sequence listing.

14) -5 Construction of 17F6 antibody heavy chain expression vector

A DNA fragment containing a nucleotide sequence encoding the 17F6 antibody heavy chain represented by SEQ ID NO: 88 of the sequence listing was synthesized (GENEART Corp. Artificial Genome Synthesis Service). 17F6 antibody heavy chain expression vector was constructed in the same manner as in Example 14) -1. The obtained expression vector was named " pCMA / 17F6H ".

The amino acid sequence of the 17F6 antibody heavy chain is shown in SEQ ID NO: 89 of the sequence listing.

14) -6 Construction of 17F6 antibody light chain expression vector

A DNA fragment containing a nucleotide sequence encoding the 17F6 antibody light chain shown in SEQ ID NO: 90 of the sequence listing was synthesized (GENEART's Strings DNA Fragments). 17F6 antibody light chain expression vector was constructed in the same manner as in Example 14) -2. The obtained expression vector was named " pCMA / 17F6L ".

The amino acid sequence of the 17F6 antibody light chain is shown in SEQ ID NO: 91 of the sequence listing.

14) -7 Preparation of 10F8 antibody, 14F74 antibody, and 17F6 antibody

14) -7-1 Production of 10F8 antibody, 14F74 antibody, and 17F6 antibody

Was produced in the same manner as in Example 12) -3-1. The 10F8 antibody was produced by the combination of pCMA / 10F8H and pCMA / 10F8L, the 14F74 antibody was produced by the combination of pCMA / 14F74H and pCMA / 14F74L, and the 17F6 antibody was produced by the combination of pCMA / 17F6H and pCMA / 17F6L .

14) -7-2 Two-step process purification of 10F8 antibody, 14F74 antibody, and 17F6 antibody

In the same manner as in Example 12) -3-2, the antibody was subjected to two-step purification of the culture supernatant obtained in Example 14) -7-1.

[Example 15] In vitro activity comparison of an effector-induced reduction variant of an affinity-matched antibody and other anti-human Orai1 antibodies

15) -1 flow cytometry of the anti-human Orai1 antibody

1) The human Orai1 expression vector constructed in -1-1 was introduced into HEK293T cells by the method shown in 1) -4-2. The cell suspension was centrifuged, and the supernatant was removed. Then, pcDNA3.1-hOrai1-introduced HEK293T cells and HEK293T cells transfected with pcDNA3.1-DEST were cultured in 2C1.1, 5H3.1, 14) -7 prepared in hR198_HG1 / LG1, hR198_HG1 / LG1-LALA, 10F8, 14F74, 17F6, and human IgG control antibody and mouse IgG control antibody as controls were added, and the mixture was allowed to stand at 4 DEG C for 30 minutes. After washing twice with PBS containing 5% FBS, the anti-human IgG FITC conjugate diluted 100-fold with PBS containing 5% FBS and mouse IgG FITC conjugate (Cappel And the mixture was allowed to stand at 4 占 폚 for 30 minutes. Washed twice with PBS containing 5% FBS, resuspended in PBS containing 5% FBS containing 1 μg / ml propidium iodide, and detection was carried out with a flow cytometer (FC500). Data analysis was performed with Flowjo. After the propidium iodide positive cells were excluded from the gates, a histogram of the FITC fluorescence intensity of the live cells was prepared and the average fluorescence intensity (MFI) was calculated. (human antibody), Fig. 11 (mouse antibody), and hR198_HG1 / LG1, hR198_HG1-LALA / LG1, 2C1.1, 5H3.1, 10F8, 14F74 and 17F6 do not bind to pcDNA3.1- , Binding to the pcDNA3.1-hOrai1-introduced HEK293T cells was observed, and it was revealed that any antibody specifically binds to human Orai1. On the other hand, no binding was observed in the mouse IgG control antibody.

15) -2 Inhibitory effect of anti-human Orai1 antibody on human T cell line activation

Human T cell line (including 10% FBS, 100 U / ㎖ penicillin and 100 ㎍ / ㎖ streptomycin), a Jurkat cell, RPMI1640 with 1.5 x 10 ⁶ cells / ㎖ was prepared at a concentration of 96-well cell culture plate And 10 μl / well of hR198_HG1 / LG1, hR198_HG1-LALA / LG1, 2C1.1, 5H3.1, 10F8, 14F74 and 17F6 were added and the cells were pretreated for 60 minutes at 37 ° C under 5% CO ₂ . Then, 100 ng / ml PMA and 1 μg / ml A23187 were added to 10 μl / well (final concentration of 10 ng / ml PMA and 100 ng / ml A23187) CO ₂ . After the plate was well stirred, it was centrifuged at 600 g for 3 minutes, and the concentration of IL-2 contained in the supernatant was measured by ELISA. Figure 12 shows that the anti-human Orai1 antibody inhibits the release of IL-2 from Jurkat cells treated with PMA and A23187 in an additive concentration-dependent manner. 13 shows the half-life inhibitory concentration (IC ₅₀ ) of hR198_HG1 / LG1, hR198_HG1-LALA / LG1, 2C1.1, 5H3.1, 10F8, 14F74 and 17F6 when the concentration of IL- And 80% inhibitory concentration (IC ₈₀ ). While the IC ₅₀ of prior art antibodies is greater than 80 ng / ml, an exemplary antibody of the invention has an IC ₅₀ of less than or equal to 10 ng / ml. Also, the IC ₈₀ of prior art antibodies is greater than 60000 ng / ml, while the IC ₈₀ of a representative antibody of the invention is less than 200 ng / ml.

15) -3 Inhibitory effect of the effector-activated variant of the affinity-matched antibody and other anti-human Orai1 antibodies on the activation of human peripheral blood mononuclear cells

Human peripheral blood mononuclear cells (PBMC) were purchased frozen from Cellular Technology and thawed according to the instructions. PBMCs prepared at a concentration of 2.0 x ¹⁰⁶ cells / ml in RPMI1640 containing 10% FBS, 100 U / ml penicillin and 100 占 퐂 / ml streptomycin were inoculated on a 96-well cell culture plate at a rate of 80 占 퐇, and various anti-human Orai1 10 [mu] l / well of the antibody was added and the mixture was pretreated for 60 minutes in a 37 [deg.] C incubator. Thereafter, 10 ng / well of 100 ng / ml PMA and 1 μg / ml of A23187 was added, well stirred, and then cultured at 37 ° C. and 5% CO _{2 for} about 16 hours. After the plates were well stirred, they were centrifuged at 600 g for 3 minutes, and the IL-2 concentration and interferon gamma (IFN-γ) concentration (MABTECH) contained in the supernatant were measured by ELISA. Figure 51 shows that the anti-human Orai1 antibody inhibits the release of IL-2 from PMA and A23187 treated human PBMC in an additive concentration-dependent manner. 52 shows the half-life inhibitory concentration (IC ₅₀ ) of hR198_HG1 / LG1, hR198_HG1-LALA / LG1, 2C1.1, 5H3.1, 10F8, 14F74 and 17F6 when the concentration of IL- And 80% inhibitory concentration (IC ₈₀ ). The IC ₅₀ of prior art antibodies is greater than 100 ng / ml, while the IC ₅₀ of a representative antibody of the invention is less than 20 ng / ml. Also, the IC ₈₀ of prior art antibodies is greater than 17000 ng / ml, while the IC ₈₀ of a representative antibody of the invention is less than 400 ng / ml. Figure 53 shows that the anti-human Orai1 antibody suppresses the release of IFN-y from human PBMCs treated with PMA and A23187 in an additive concentration-dependent manner. 54 shows the half-life inhibitory concentration (IC ₅₀ ) of hR198_HG1 / LG1, hR198_HG1-LALA / LG1, 2C1.1, 5H3.1, 10F8, 14F74 and 17F6, when the concentration of IFN- And 80% inhibitory concentration (IC ₈₀ ). The IC ₅₀ of prior art antibodies is greater than 800 ng / ml, while the IC ₅₀ of a representative antibody of the invention is 40 ng / ml or less. Also, the IC ₈₀ of prior art antibodies is greater than 300000 ng / ml, while the IC ₈₀ of a representative antibody of the invention is less than 2000 ng / ml.

[Example 16] In vivo activity of hR198_HG1 / LG1

16) -1 administration effect of hR198_HG1 / LG1 on human graft versus host disease model

It has been known that human graft-versus-host-like responses can be induced by introducing human PBMC into NSG mice (NOD. Cg-Prkdc <scid> Il2rg <tm1Wjl> / SzJ), a severe combined immunodeficient mouse Experimental Immunology, 157: 104-118 (2009)). Seventeen male six-week old NSG mice purchased from Nippon Charles River Company were irradiated with 2.0 Gy of X-ray (Hitachi X-ray Irradiation Apparatus MBR-1520R-4), and mice were divided into two groups of 1 and 5 . And 30 mg / kg and 60 mg / kg of hR198_HG1 / LG1 prepared to be 3 mg / ml and 6 mg / ml with HBSor (25 mM hystidine / 5% sorbitol, pH 6.0) n = 5) mice intraperitoneally. Group 1 (n = 5) was administered only HBSor as a vehicle group. On the next day, freezing human PBMC (Cellular Technology) was thawed according to the protocol using CTL-antiaggrigate (Cellular Technology), and 3 million human PBMCs were suspended in 200 μl of PBS to give 3 groups (n = 5) , And group 1 (n = 2) was observed as progression as an X-ray irradiation control group without introducing human PBMC. In the administration group, the same dose of hR198_HG1 / LG1 or HBSor was administered at days 0, 7, 14 and 21 after X irradiation. The weight of each mouse was measured every day after Day 0, Day 1, Day 4, Day 7, Day 9, Day 10, Day 11 and Day 13. The body weight of Day 0 was 100% The average weight change of each group is shown in Fig. The decrease in the mean body weight of the vehicle-treated group began to be observed from the 16th day. The experiment was completed on day 21 when the average weight of the vehicle-administered group was less than 80%. At this point, the X-ray irradiation control group without the introduction of human PBMC showed no weight loss. Mice receiving hR198_HG1 / LG1 at 30 mg / kg and 60 mg / kg did not decrease in body weight, and the mean body weight was equivalent to that of human PBMC-uninfected mice. In this system, hR198_HG1 / LG1 (anti-Orai1 antibody), which significantly suppresses the symptoms of graft-versus host disease by activation of human PBMC, is expected to have therapeutic and / or preventive effects on human graft versus host disease.

16) -2 Evaluation of in vivo activity of hR198_HG1 / LG1 using human Orai1 knock mouse

16) -2-1 Production of human Orai1 knock-in mouse

A human Orai1 knock mouse in which the amino acid sequence of the second extracellular loop domain of mouse Orai1 was replaced with a human Orai1 sequence was produced in Tokushu Immunization Laboratory Co., Ltd. according to the method of genetically modified mouse production. In summary, the DNA sequence of the coding region of the BAC clone containing the mouse Orai1 locus was replaced with the human Orai1 locus, and the neo resistance gene inserted in the loxP sequence was introduced to introduce the knocking targeting vector . This targeting vector was introduced into mouse ES cells to establish G418 resistance. By the Southern hybridization method, an ES cell line specifically reassembled the target locus was screened. Further, a Cre expression vector was introduced to remove the selection marker, and a chimeric F1 mouse was produced using the ES cell line. Genotyping was performed by Southern hybridization to select heterozygous individuals among the F1 mice, and by crossing the F1 heterozygotes, a human Orai1 knock mutant, F2 homozygous mutant, was generated.

16) -2-2 Inhibitory effect of hR198_HG1 / LG1 on passive skin anaphylaxis (PCA)

The mouse PCA reaction was performed according to a regular method. 8-week-old human Orai1 knock mice or wild-type co-broth mice produced by the Tokushu Immunization Laboratory were calibrated with a calibrator and 10 ml / kg of hR198_HG1 / LG1 prepared to be 6 mg / ml in HBSor, that is, 60 mg / Were administered intravenously to the tail. In the vehicle group, only HBSor was administered. On the following day, 10 μl of monoclonal anti-OVA IgE (chondrex), adjusted to 10 μg / ml with physiological saline, was administered intrathecally under isoflurane (phytoplankton) inhalation anesthesia. After 24 hours, physiological saline containing 2 mg / ml of OVA (Albumin from chicken egg white, SIGMA) and 20 mg / ml of Evans blue (Merck) was administered at 5 mg / kg OVA and 100 Mg / kg Evans blue. &Lt; / RTI &gt; After 30 minutes, the isoflurane was removed from the corpuscle, and the auricle was excised and immersed in 0.5 ml of DMSO to extract Evans blue (37 ° C, 72 hours). The DMSO solution from which Evans blue was extracted was transferred to a 96-well microplate in 200 쨉 l increments, 650 nm was measured with a microplate reader (Molecular devices, SpectraMax M5e). The absorbance of the leavened Evans blue was calculated by the following calculation, taking the average value of the vehicle-treated group as 100%. In Blank, the absorbance of the DMSO solution was determined. % = (OD650 sample - OD650 blank) / (OD650 vehicle - OD650 blank). FIG. 56 shows that hR198_HG1 / LG1 inhibits the PCA reaction induced in mice that are human Orai1 knock. The mouse PCA response is a basic model system of immediate allergy in which anaphylactic reactions of the human type I allergic reaction are reproduced and it is known that this system can be used for the evaluation of antiallergic drugs (Archives internationals de pharmacodynamie et de therapie, 165: 102 (1967), International Archives of Allergy and Applied Immunology, 78: 113-117 (1985)). In this system, hR198_HG1 / LG1 (anti-Orai1 antibody), which has been shown to inhibit IgE-dependent mast cell degranulation, has been shown to inhibit bronchial asthma, allergic rhinitis, atopic dermatitis, Also, treatment and / or prophylactic effects of the same human type I allergic diseases, such as allergic asthma, etc., are expected.

16) -2-3 Inhibitory effect of hR198_HG1 / LG1 on delayed hypersensitivity (DTH)

The mouse DTH reaction was carried out according to the conventional method. 8-week-old human Orai1 knock mice or wild-type co-broth mice produced by the Tokushu Immunization Laboratory were calibrated with a calibrator and 10 ml / kg of hR198_HG1 / LG1 prepared to be 6 mg / ml in HBSor, that is, 60 mg / Were administered intravenously to the tail. In the vehicle group, only HBSor was administered. The next day, 50 μl of the emulsion prepared by mixing equal amounts of mBSA (Albumin Bovine Methylated, SIGMA) and Freund's Complete Adjuvant (DIFCO) diluted to 5 mg / ml with physiological saline was administered subcutaneously under both solutions to perform immunization. After 6 days, hR198_HG1 / LG1 was administered intravenously to the tail vein at 60 mg / kg. The following day, mBSA and physiological saline prepared with 0.5 mg / ml of physiological saline were administered intradermally to one leg under isoflurane inhalation anesthesia, respectively, and swelling of the legs after 6, 24 and 48 hours after administration were measured by a dial gauge. The swelling of the legs was 0 (10 ^-2 mm) at 0 hour, and the thickness of the swelling over time was calculated, and the average value of the vehicle-treated group was taken as 100%. FIG. 57 shows that the DTH reaction induced in the human Orai1 knock mouse was inhibited by hR198_HG1 / LG1 at 6 hours (A) 24 hours (B) 48 hours (C) after the antigen administration. It is well known that the mouse DTH response is a system that observes T cell dependent immune establishment and response, and that agents exhibiting activity in this system have been developed as powerful immunosuppressive agents (Clinical and Experimental Immunology, 52: 599-606 )). HR198_HG1 / LG1 (anti-Orai1 antibody), which has been shown to inhibit T cell immunity in this system, has been shown to be effective against human vital reactions or diseases caused by T cell activity, such as rejection of grafted immune diseases, A therapeutic and / or preventive effect is expected.

16) -2-4 Review of inhibitory activity of Orai1 antibody against dermatitis

Dermatitis is caused by any of the following methods. 0.5 ml of albumin solution was intraperitoneally injected into the abdominal cavity of the mice and further immunized with the same amount of antigen two weeks later. The albumin antiserum was added to the ear (20 μl) or the aliquot (100 μl) every 3 days to 2 weeks Repeat 3-6 times. Alternatively, the mite antigen cream is repeatedly applied to the ear (20 μl) or the aliquot (100 μl) 3 to 6 times every 3 to 2 weeks. Or hindered picryl chloride or dinitrofluorobenzene is prepared according to the protocol and applied to the ear (20 μl) or aliquot (100 μl) once or twice weekly for up to 8 weeks. Histamine, Compound 40/80, 5- (and 6-) carboxyfluorescein diacetate succinimidyl ester, 5- (and 6-) carboxyfluorescein diacetate succinimidyl ester, Fluorescein isothiocyanate and Bombesin-like peptide are administered to the ear (20 μl), aliquot (100 μl), and spinal cord (5 μl). The anti-Orai1 antibody is administered intravenously or subcutaneously on the day before or 1 to 4 hours before induction of dermatitis. Thereafter, the administration of the antibody is continued at an interval of 7 to 28 days. After inducing dermatitis, measure the thickness of the auricle using a dialysis gauge over time, and perform gross scoring of dermatitis. After completion of the test period, quantification of antibody, cytokine, blood biomarker concentration in blood or tissue, cell proliferation activity and cytokine production ability from skin, peripheral blood, thymus, spleen, lymph node and bone marrow, Examination, pathological tissue analysis, and the like to determine the inhibitory activity of the anti-Orai1 antibody against dermatitis.

16) -2-5 Review of the inhibitory activity of Orai1 antibody against psoriasis

When imiquimod is used, it is applied to both sides of the auricle or one side (5 ~ 30 ㎎) and a part (50 ~ 100 ㎎) in which hairs are completed to induce the dermatitis. Or a suspension of 10 mg / ml of Zymosan phosphate buffer is intraperitoneally administered to induce dermatitis. In the case of using cytokine (IL-23 or the like) as a base substance, 20 to 50 μl of a solution containing 0.1 to 2 μg of cytokine is injected intradermally on the surface of mouse ear canal under 1 to 5% isoflurane anesthesia Induces a dry skin dermatitis. The anti-Orai1 antibody is administered intravenously or subcutaneously on the day before or 1 to 4 hours before induction of dermatitis. Thereafter, the administration of the antibody is continued at an interval of 7 to 28 days. After inducing dermatitis, measure the thickness of the auricle using a dialysis gauge over time, and perform a gross scoring of dermatitis. After the end of the test period, the weight of the inflammation site and the myeloperoxidase activity of neutrophils infiltrating the site, the flow cytometry analysis of infiltrated cells, the gene analysis, the cytokine concentration measurement, Activity is judged.

16) -2-6 Review of inhibitory activity of Orai1 antibody against multiple sclerosis

Myelin oligodendrocyte glycoprotein or its peptide antigen was adjusted to 4 mg / ml with physiological saline and mixed with an equivalent amount of Freund's complete adjuvant adjusted to 8 mg / ml with physiological saline, 200 μl of this mixed solution is administered to the side of the mouse or to the abdomen of the abdomen. Immediately thereafter, 2 μg / ml of pertussis toxin aqueous solution is administered to the same mouse from 100 μl of the tail vein. After 2 days, the pertussis toxin solution described above is again administered from the tail vein to induce experimental encephalomyelitis. From 1 week to 2 weeks after the initiation of the experiment, encephalomyelitis develops and the paralysis extends from the tail to the lower limbs and the forelimbs. The degree of this paralysis is scored by observing the movements of limbs and tails grossly. The anti-Orai1 antibody is administered intravenously or subcutaneously on the day before or 1 to 4 hours before induction of encephalomyelitis. Thereafter, the administration of the antibody is continued at an interval of 7 to 28 days, and the administration effect on the multiple sclerosis of the anti-Orai1 antibody is determined.

16) -2-7 Review of the inhibitory activity of Orai1 antibody against arthritis

2 ㎎ / ㎖ of bovine type II collagen and complete adjuvant of Freund are mixed at a volume ratio of 1: 1.3 and emulsified. 100 쨉 l of the solution is injected into the myotubes of the mice using 1 ml of injected tuberculin needle. Two to three weeks later, the same treatment is performed, and the subsequent joint swelling of the extremities is scored using a glance or a dialysis gauge. At the end of the test period, the antibody, cytokine, blood biomarker concentration in blood or tissue, cell proliferation activity, cytokine production ability, surface antigen, etc. obtained from skin, peripheral blood, thymus, spleen, lymph node and bone marrow are measured. The anti-Orai1 antibody is administered intravenously or subcutaneously on the day before or 1 to 4 hours before induction of arthritis. Thereafter, the administration of the antibody is continued at an interval of 7 to 28 days, and the administration effect of the anti-Orai1 antibody against arthritis is determined.

16) -2-8 Review of inhibitory activity of Orai1 antibody against colitis

The lymphocytes collected and purified from lymph nodes and spleen of human Orai1 knock mice were isolated by cell sorter using anti-CD4 antibody GK1.5, anti-CD25 antibody PC61.5, anti-CD45R antibody C363.16A (all from eBioscience) do. After confirming that the cells obtained by this method are CD4 + CD25-CD45RBhi T-cells having a purity of 95% or more by a flow cytometer, 0.5 million of them are transferred into the abdominal cavity of Rag2 - / - mice at 12-16 weeks of age. After 12 weeks, we observe symptoms such as weight measurement and diarrhea. After the observation period, we examine the enlargement of the intestinal tract by the autopsy, the number and size of polyps, and the presence of pathological features. Quantification of antibody, cytokine, and biomarker concentration in blood or tissue, cell proliferation activity, cytokine production ability, surface antigen, etc. obtained from the intestinal, peripheral blood, thymus, spleen, lymph node, and bone marrow are analyzed. The anti-Orai1 antibody is administered intravenously or subcutaneously on the day before or 1 to 4 hours before cell entry. Thereafter, administration of the antibody is continued at an interval of 7 to 28 days, and the administration effect of the anti-Orai1 antibody against colitis is judged.

16) -2-9 Review of the effect of Orai1 antibody on bone marrow cell transplantation system

The femur and tibia of a human Orai1 knock mouse were extracted and the cells in the bone marrow were extruded by injecting 1 ~ 5 ml of 10% FBS-RPMI1640 medium from the neck using a needle syringe and centrifugation after cell strainer treatment . Bone marrow cells were collected, and 10 mM HEPES, 0.5 mM EDTA, and 0.5% penicillin / streptomycin were added to the injection buffer to adjust 10 million bone marrow cells out of 200 μl. At the same time, non-cells are collected from the spleen of a human Orai1 knock mouse according to the conventional method. BALB / c mice of 10 to 13 weeks of age were irradiated with 2.0 to 8.0 Gy of X-rays, and then 0 to 4 million donor splenocytes were added to 200 μl of donor bone marrow cells. The cell mixture was added to the recipient Inject from the tail vein. Thereafter, the body weight change or survival rate of 4 to 16 weeks, the concentration of antibodies, cytokines, blood biomarkers in the blood or tissues of recipient mice at the end of the experiment, and the number of cells obtained from the liver, peripheral blood, thymus, spleen, lymph node, Surface antigen, proliferative activity, cytokine production ability is measured. The anti-Orai1 antibody is administered to the recipient mice intravenously or subcutaneously on the day before or 1 to 4 hours before the bone marrow transplantation. Thereafter, the administration of the antibody is continued at an interval of 7 to 28 days, and the action of the anti-Orai1 antibody on the engraftment of the grafted cells or the incidence of graft-versus-host disease is determined.

[Example 17] Comparison of physicochemical properties between an affinity-matched antibody and other anti-human Orai1 antibodies

Viscosity measurement by m-VROC

1) produced by hR198_HG1-LALA / LG1 and 13) -6 produced in hR198_HG1 / LG1, hR198_H3 / LG1, / Ml, and then adjusted to 90, 120, and 150 mg / ml in HBSor. The viscosity at 25 캜 was measured three times at each concentration using m-VROC (RheoSense Co.), and the average viscosity (mPa s) was calculated. The results are shown in Fig. 58 and Table 1. The viscosity of hR198_HG1 / LG1, hR198_H3 / LG1 and hR198_HG1-LALA / LG1 was lower than that of 2C1.1 at any concentration. In particular, at 150 mg / ml, the viscosities of hR198_HG1 / LG1, hR198_H3 / LG1 and hR198_HG1-LALA / LG1 were 11.4, 10.0 and 10.8 mPa · s respectively, whereas 2C1.1 was 21.0 mPa · s. 1, the affinity-matched antibody exhibited a low viscosity even when it was highly concentrated.

Industrial availability

The humanized anti-Orai1 antibody of the present invention has a more potent T cell activity inhibitory action than known antibodies, and thus can be a therapeutic or preventive agent such as transplant rejection and the like.

Array table free text

SEQ ID NO: 1: Gene sequence of human Orai1

SEQ ID NO: 2: Amino acid sequence of human Orai1

SEQ ID NO: 3: PCR primer Nhe-polyC-S

SEQ ID NO: 4: PCR primer rIgγ-AS1

SEQ ID NO: 5: PCR primer rIg gamma-AS2

SEQ ID NO: 6: PCR primer Nhe-polyC-S

SEQ ID NO: 7: PCR primer rIgκ-AS

SEQ ID NO: 8 Sequence primer rIg? -Seq

SEQ ID NO: 9: Sequence primer rIgκ-seq

SEQ ID NO: 10: R118 Nucleotide sequence encoding light chain

SEQ ID NO: 11: R118 amino acid sequence of light chain

SEQ ID NO: 12: R118 Nucleotide sequence encoding heavy chain

SEQ ID NO: 13: R118 amino acid sequence of the heavy chain

SEQ ID NO: 14: Nucleotide sequence encoding the light chain of R198

SEQ ID NO: 15: R198 amino acid sequence of light chain

SEQ ID NO: 16: R198 Nucleotide sequence encoding heavy chain

SEQ ID NO: 17: R198 amino acid sequence of the heavy chain

SEQ ID NO: 18: DNA fragment containing sequence encoding human κ chain secretion signal and human κ chain normal region

SEQ ID NO: 19: PCR primer 3.3-F1

SEQ ID NO: 20: PCR primer 3.3-R1

SEQ ID NO: 21: A DNA fragment comprising the sequence of the human midgamer signal sequence and the amino acid sequence of the human IgG1 normal region

SEQ ID NO: 22: Nucleotide sequence encoding human chimeric cR118 light chain

SEQ ID NO: 23: Amino acid sequence of human chimeric lysine cR118 light chain

SEQ ID NO: 24: Nucleotide sequence encoding human chimeric cR198 light chain

SEQ ID NO: 25: amino acid sequence of human chimeric lysine cR198 light chain

SEQ ID NO: 26: Nucleotide sequence encoding human chimeric cR118 heavy chain

SEQ ID NO: 27: amino acid sequence of human chimeric cR118 heavy chain

SEQ ID NO: 28: Nucleotide sequence encoding human chimeric cR198 heavy chain

SEQ ID NO: 29: amino acid sequence of human chimerized cR198 heavy chain

SEQ ID NO: 30: nucleotide sequence encoding hR198_L1 type light chain

SEQ ID NO: 31: amino acid sequence of hR198_L1 type light chain

SEQ ID NO: 32: nucleotide sequence encoding hR198_L2 type light chain

SEQ ID NO: 33: amino acid sequence of hR198_L2 type light chain

SEQ ID NO: 34: nucleotide sequence encoding hR198_L3 type light chain

SEQ ID NO: 35: amino acid sequence of hR198_L3 type light chain

SEQ ID NO: 36: nucleotide sequence encoding hR198_L4 type light chain

SEQ ID NO: 37: amino acid sequence of hR198_L4 type light chain

SEQ ID NO: 38: nucleotide sequence encoding hR198_H1 type heavy chain

SEQ ID NO: 39: amino acid sequence of hR198_H1 type heavy chain

SEQ ID NO: 40: nucleotide sequence encoding hR198_H2 type heavy chain

SEQ ID NO: 41: amino acid sequence of hR198_H2 type heavy chain

SEQ ID NO: 42: nucleotide sequence encoding hR198_H3 type heavy chain

SEQ ID NO: 43: amino acid sequence of hR198_H3 type heavy chain

SEQ ID NO: 44: nucleotide sequence encoding the hR198_H4 type heavy chain

SEQ ID NO: 45: amino acid sequence of hR198_H4 type heavy chain

SEQ ID NO: 46: PCR primer Orai1 HF

SEQ ID NO: 47: PCR primer Orai1 CH FR

SEQ ID NO: 48: PCR primer M13 rev long

SEQ ID NO: 49: PCR primer SecM Stop R

SEQ ID NO: 50: PCR primer Orai1 Lc F

SEQ ID NO: 51: PCR primer Orai1 CL-FR

SEQ ID NO: 52: PCR primer Orai1 HR-FLAG R

SEQ ID NO: 53: PCR primer Orai1 CL-FLAG R

SEQ ID NO: 54: PCR primer Myc-R

SEQ ID NO: 55: nucleotide sequence encoding hR198_LG1 type light chain

SEQ ID NO: 56: amino acid sequence of hR198_LG1 type light chain

SEQ ID NO: 57: Nucleotide sequence encoding hR198_LG2 type light chain

SEQ ID NO: 58: amino acid sequence of hR198_LG2 type light chain

SEQ ID NO: 59: nucleotide sequence encoding hR198_LG3 type light chain

SEQ ID NO: 60: amino acid sequence of light chain of hR198_LG3 type

SEQ ID NO: 61: nucleotide sequence encoding hR198_HG1 type heavy chain

SEQ ID NO: 62: amino acid sequence of hR198_HG1 type heavy chain

SEQ ID NO: 63: nucleotide sequence encoding the hR198_HG2 type heavy chain

SEQ ID NO: 64: amino acid sequence of hR198_HG2 type heavy chain

SEQ ID NO: 65: nucleotide sequence encoding the hR198_HG3 type heavy chain

SEQ ID NO: 66: amino acid sequence of hR198_HG3 type heavy chain

SEQ ID NO: 67: nucleotide sequence encoding hR198_H4-LALA type heavy chain

SEQ ID NO: 68: hR198_H4-amino acid sequence of LALA type heavy chain

SEQ ID NO: 69: hR198_HG1-Nucleotide sequence encoding LALA type heavy chain

SEQ ID NO: 70: hR198_HG1-amino acid sequence of LALA type heavy chain

SEQ ID NO: 71: A DNA fragment comprising an amino acid sequence encoding a human midgut secretion signal and a human IgG2 normal region

SEQ ID NO: 72: 2C1.1 Nucleotide sequence encoding antibody heavy chain

SEQ ID NO: 73: 2C1.1 amino acid sequence of antibody heavy chain

SEQ ID NO: 74: 2C1.1 Nucleotide sequence encoding antibody light chain

SEQ ID NO: 75: 2C1.1 amino acid sequence of antibody light chain

SEQ ID NO: 76: 5H3.1 Nucleotide sequence encoding antibody heavy chain

SEQ ID NO: 77: Amino acid sequence of 5H3.1 antibody heavy chain

SEQ ID NO: 78: 5H3.1 Nucleotide sequence encoding light chain of antibody

SEQ ID NO: 79: 5H3.1 amino acid sequence of antibody light chain

SEQ ID NO: 80: Nucleotide sequence encoding 10F8 antibody heavy chain

SEQ ID NO: 81: amino acid sequence of 10F8 antibody heavy chain

SEQ ID NO: 82: Nucleotide sequence encoding the 10F8 antibody light chain

SEQ ID NO: 83: 10F8 amino acid sequence of antibody light chain

SEQ ID NO: 84: Nucleotide sequence encoding the antibody heavy chain

SEQ ID NO: 85: amino acid sequence of the 14F74 antibody heavy chain

SEQ ID NO: 86: Nucleotide sequence encoding antibody light chain

SEQ ID NO: 87: 14F74 amino acid sequence of antibody light chain

SEQ ID NO: 88: 17F6 Antibody Nucleotide sequence encoding heavy chain

SEQ ID NO: 89: The amino acid sequence of the 17F6 antibody heavy chain

SEQ ID NO: 90: Nucleotide sequence encoding 17F6 antibody light chain

SEQ ID NO: 91: amino acid sequence of 17F6 antibody light chain

SEQ ID NO: 92: Rat CDRL1 (derived from R198, used for hR198_L1 to L4 type light chain)

SEQ ID NO: 93: variant CDRL1 (used for hR198_LG1 type light chain)

SEQ ID NO: 94: variant CDRL1 (used for hR198_LG2 type light chain)

SEQ ID NO: 95: variant CDRL1 (used for hR198_LG3 type light chain)

SEQ ID NO: 96: rat CDRL2 (common in R118 and R198, used in hR198_L1 to L4 and LG1 type light chains)

SEQ ID NO: 97: variant CDRL2 (used for hR198_LG2 type light chain)

SEQ ID NO: 98: variant CDRL2 (used for hR198_LG3 type light chain)

SEQ ID NO: 99: Rat CDRL3 (derived from R118, used for hR198_L3 and L4 type light chains)

SEQ ID NO: 100: Rat CDRL3 (derived from R198, used for hR198_L1 and L2 type light chains)

SEQ ID NO: 101: variant CDRL3 (used for hR198_LG1 to LG3 type light chain)

SEQ ID NO: 102: Rat CDRH1 (derived from R118, used for hR198_H3, H4 and HG1 to HG4 type heavy chain)

SEQ ID NO: 103: Rat CDRH1 (derived from R198, used for hR198_H1 and H2 type heavy chain)

SEQ ID NO: 104: Rat CDRH2 (derived from R118, used for hR198_H3 and H4 type heavy chain)

SEQ ID NO: 105: Rat CDRH2 (derived from R198, used for hR198_H1 and H2 type heavy chain)

SEQ ID NO: 106: Variation CDRH2 (used for hR198_HG1 type heavy chain)

SEQ ID NO: 107: Variation CDRH2 (used for hR198_HG2 type heavy chain)

SEQ ID NO: 108: variant CDRH2 (used for hR198_HG3 type heavy chain)

SEQ ID NO: 109: rat CDRH3 (common for R118 and R198, used for hR198_H1 to H4 type heavy chain)

SEQ ID NO: 110: variant CDRH3 (used for hR198_HG1 to HG3 type heavy chain)

SEQ ID NO: 111: nucleotide sequence encoding hR198_H0 type heavy chain

SEQ ID NO: 112: amino acid sequence of hR198_H0 type heavy chain

SEQ ID NO: 113: nucleotide sequence encoding hR198_H5 type heavy chain

SEQ ID NO: 114: amino acid sequence of hR198_H5 type heavy chain

SEQ ID NO: 115: Biotin PEGylated human Orai1 loop region peptide sequence

SEQ ID NO: 116: rat CDRL1 (derived from R118, but not used in humanized antibodies)

                         SEQUENCE LISTING &Lt; 110 > Daiichi Sankyo Co., Ltd.   <120> Anti Orai1 antibodies <130> FP1522 <160> 116 <170> PatentIn version 3.5 <210> 1 <211> 906 <212> DNA <213> Homo sapiens <400> 1 atgcatccgg agcccgcccc gcccccgagc cgtagcagtc ccgagcttcc cccaagcggc 60 ggcagcacca ccagcggcag ccgccggagc cgccgccgca gcggggacgg ggagcccccg 120 ggggccccgc caccgccgcc gtccgccgtc acctacccgg actggatcgg ccagagttac 180 tccgaggtga tgagcctcaa cgagcactcc atgcaggcgc tgtcctggcg caagctctac 240 ttgagccgcg ccaagcttaa agcctccagc cggacctcgg ctctgctctc cggcttcgcc 300 atggtggcaa tggtggaggt gcagctggac gctgaccacg actacccacc ggggctgctc 360 atcgccttca gtgcctgcac cacagtgctg gtggctgtgc acctgtttgc gctcatgatc 420 agcacctgca tcctgcccaa catcgaggcg gtgagcaacg tgcacaatct caactcggtc 480 aaggagtccc cccatgagcg catgcaccgc cacatcgagc tggcctgggc cttctccacc 540 gtcattggca cgctgctctt cctagctgag gtggtgctgc tctgctgggt caagttcttg 600 cccctcaaga agcagccagg ccagccaagg cccaccagca agccccccgc cagtggcgca 660 gcagccaacg tcagcaccag cggcatcacc ccgggccagg cagctgccat cgcctcgacc 720 accatcatgg tgcccttcgg cctgatcttt atcgtcttcg ccgtccactt ctaccgctca 780 ctggttagcc ataagaccga ccgacagttc caggagctca acgagctggc ggagtttgcc 840 cgcttacagg accagctgga ccacagaggg gaccaccccc tgacgcccgg cagccactat 900 gcctag 906 <210> 2 <211> 301 <212> PRT <213> Homo sapiens <400> 2 Met His Pro Glu Pro Ala Pro Pro Pro Ser Arg Ser Ser Pro Glu Leu 1 5 10 15 Pro Pro Ser Gly Gly Ser Thr Thr Ser Gly Ser Arg Arg Ser Ser Arg             20 25 30 Arg Ser Gly Asp Gly Glu Pro Pro Gly Ala Pro Pro Pro Pro Ser         35 40 45 Ala Val Thr Tyr Pro Asp Trp Ile Gly Gln Ser Tyr Ser Glu Val Met     50 55 60 Ser Leu Asn Glu His Ser Met Gln Ala Leu Ser Trp Arg Lys Leu Tyr 65 70 75 80 Leu Ser Arg Ala Lys Leu Lys Ala Ser Ser Arg Thr Ser Ala Leu Leu                 85 90 95 Ser Gly Phe Ala Met Val Ala Met Val Glu Val Gln Leu Asp Ala Asp             100 105 110 His Asp Tyr Pro Pro Gly Leu Leu Ile Ala Phe Ser Ala Cys Thr Thr         115 120 125 Val Leu Val Ala Val Le Le Phe Ala Leu Met Ile Ser Thr Cys Ile     130 135 140 Leu Pro Asn Ile Glu Ala Val Ser Asn Val His Asn Leu Asn Ser Val 145 150 155 160 Lys Glu Ser Pro His Glu Arg Met His Arg His His Glu Leu Ala Trp                 165 170 175 Ala Phe Ser Thr Val Ile Gly Thr Leu Leu Phe Leu Ala Glu Val Val             180 185 190 Leu Leu Cys Trp Val Lys Phe Leu Pro Leu Lys Lys Gln Pro Gly Gln         195 200 205 Pro Arg Pro Thr Ser Lys Pro Pro Ala Ser Gly Ala Ala Ala Asn Val     210 215 220 Ser Thr Ser Gly Ile Thr Pro Gly Gln Ala Ala Ile Ala Ser Thr 225 230 235 240 Thr Ile Met Val Pro Phe Gly Leu Ile Phe Ile Val Phe Ala Val His                 245 250 255 Phe Tyr Arg Ser Leu Val Ser His Lys Thr Asp Arg Gln Phe Gln Glu             260 265 270 Leu Asn Glu Leu Ala Glu Phe Ala Arg Leu Gln Asp Gln Leu Asp His         275 280 285 Arg Gly Asp His Pro Leu Thr Pro Gly Ser His Tyr Ala     290 295 300 <210> 3 <211> 37 <212> DNA <213> artificial <220> PCR Primer Nhe-polyC-S <220> <221> misc_feature <222> (37). (37) <223> n is a, c, g, or t <400> 3 gctagcgcta ccggactcag atcccccccc cccccdn 37 <210> 4 <211> 28 <212> DNA <213> artificial <220> <223> PCR Primer rIg gamma-AS1 <400> 4 tcactgagct ggtgagagtg tagagccc 28 <210> 5 <211> 28 <212> DNA <213> artificial <220> <223> PCR Primer rIG gamma-AS2 <400> 5 tcaccgagct gctgagggtg tagagccc 28 <210> 6 <211> 37 <212> DNA <213> artificial <220> PCR Primer Nhe-polyC-S <220> <221> misc_feature <222> (37). (37) <223> n is a, c, g, or t <400> 6 gctagcgcta ccggactcag atcccccccc cccccdn 37 <210> 7 <211> 28 <212> DNA <213> artificial <220> &Lt; 223 > rIg kappa-AS <400> 7 tcagtaacac tgtccaggac accatctc 28 <210> 8 <211> 19 <212> DNA <213> artificial <220> <223> rIG gamma-seq <400> 8 ctggctcagg gaaatagcc 19 <210> 9 <211> 19 <212> DNA <213> artificial <220> <223> rIG kappa-seq <400> 9 tccagttgct aactgttcc 19 <210> 10 <211> 405 <212> DNA <213> Rattus rattus <220> <221> misc_feature <222> (382). (382) <223> n is a, c, g, or t <220> <221> misc_feature &Lt; 222 > (403) .. (403) <223> n is a, c, g, or t <400> 10 atgaaaatga cgacacctgc tcagttcctt gggcttctgt tgctctggtt tccaggtgcc 60 aggtgtgaca tccagttgac ccagtctcca tccacattgc ctgcatccct gggagagaga 120 gtcaccatca gttgcagagc aagtcagagt attagcaata gtttaagctg gtttcaacag 180 aaaccagatg gaactgttaa acgcctgatc tattctacat ccactttaga atctggtgtc 240 ccatcaaggt tcagtggcag tgggtctggg acagattatt ctctctccat caccagtctt 300 gagtctgaag attttgcaat gtattactgt ctacagtttg ctacttttcc ggacacgttt 360 ggaactggga ccaaactgga antgagacgg gctgatgctg canca 405 <210> 11 <211> 135 <212> PRT <213> Rattus rattus <220> <221> misc_feature &Lt; 222 > (128) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <135> (135) <223> Xaa can be any naturally occurring amino acid <400> 11 Met Lys Met Thr Thr Pro Ala Gln Phe Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Phe Pro Gly Ala Arg Cys Asp Ile Gln Leu Thr Gln Ser Ser Ser Thr             20 25 30 Leu Pro Ala Ser Leu Gly Glu Arg Val Thr Ile Ser Cys Arg Ala Ser         35 40 45 Gln Ser Ile Ser Asn Ser Leu Ser Trp Phe Gln Gln Lys Pro Asp Gly     50 55 60 Thr Val Lys Arg Leu Ile Tyr Ser Thr Ser Thr Leu Glu Ser Gly Val 65 70 75 80 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Ser                 85 90 95 Ile Thr Ser Leu Glu Ser Glu Asp Phe Ala Met Tyr Tyr Cys Leu Gln             100 105 110 Phe Ala Thr Phe Pro Asp Thr Phe Gly Thr Gly Thr Lys Leu Glu Xaa         115 120 125 Arg Arg Ala Asp Ala Ala Xaa     130 135 <210> 12 <211> 468 <212> DNA <213> Rattus rattus <220> <221> misc_feature <222> (467). (467) <223> n is a, c, g, or t <400> 12 atggaatgga actgggtctt tctcctcctc ctgtcagtaa ctgcagaagt ccagtcccag 60 gtccagctgc agcagtctgg agcggagctg gcaaagcctg gctcctcagt gaagatttcc 120 tgcaaggctt ccggctacac cgtcaccgcc tattatataa gttggataag gcagacgatt 180 ggacagggcc ttgagtatgt tggatatatt gacatgggaa atggaaggac taactacaat 240 gcgaggttca agggcaaggc cacattgact gtggacaaat cctccagcac agccttcatg 300 caactcagca gcctgacacc tgacgactct gcggtctatt actgtgcaag ggactccaac 360 tggggggttg attactgggg ccaaggagtc atggtcacag tctcctcagc tgaaacaaca 420 gccccatctg tctatccact ggctcctgga actgcttctc aaaagtna 468 <210> 13 <211> 156 <212> PRT <213> Rattus rattus <220> <221> misc_feature <156> (156) <223> Xaa can be any naturally occurring amino acid <400> 13 Met Glu Trp Asn Trp Val Phe Leu Leu Leu Leu Ser Val Thr Ala Glu 1 5 10 15 Val Gln Ser Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Lys             20 25 30 Pro Gly Ser Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Val         35 40 45 Thr Ala Tyr Ile Ser Trp Ile Arg Gln Thr Ile Gly Gln Gly Leu     50 55 60 Glu Tyr Val Gly Tyr Ile Asp Met Gly Asn Gly Arg Thr Asn Tyr Asn 65 70 75 80 Ala Arg Phe Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser                 85 90 95 Thr Ala Phe Met Gln Leu Ser Ser Leu Thr Pro Asp Asp Ser Ala Val             100 105 110 Tyr Tyr Cys Ala Arg Asp Ser Asn Trp Gly Val Asp Tyr Trp Gly Gln         115 120 125 Gly Val Met Val Thr Val Ser Ser Ala Glu Thr Ala Pro Ser Val     130 135 140 Tyr Pro Leu Ala Pro Gly Thr Ala Ser Gln Lys Xaa 145 150 155 <210> 14 <211> 408 <212> DNA <213> Rattus rattus <220> <221> misc_feature &Lt; 222 > (403) .. (403) <223> n is a, c, g, or t <400> 14 atgaaaatga cgacacctgc tcagttcctt gggcttctgt tgctctggtt tccaggtgcc 60 aggtgtgaca tccagttgac ccagtctcca tccacattgc ctgcatctct gggagagaga 120 gtcaccatca gttgcagagc aagtcagagt attggcaata gtttaagctg gtttcagcag 180 aaaccagatg gatctgttaa acgcctgatc tactctacat ccactttaga atctggtgtc 240 ccatcaaggt tcagtggcag tgggtctggg acagattatt ctctctccat caccagtctt 300 gagtctgaag attttgcaat gtattactgt ctacagtttg ctacttatcc ggacacgttt 360 ggaactggga ccaaactgga actgagacgg gctgatgctg cancaact 408 <210> 15 <211> 136 <212> PRT <213> Rattus rattus <220> <221> misc_feature <135> (135) <223> Xaa can be any naturally occurring amino acid <400> 15 Met Lys Met Thr Thr Pro Ala Gln Phe Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Phe Pro Gly Ala Arg Cys Asp Ile Gln Leu Thr Gln Ser Ser Ser Thr             20 25 30 Leu Pro Ala Ser Leu Gly Glu Arg Val Thr Ile Ser Cys Arg Ala Ser         35 40 45 Gln Ser Ile Gly Asn Ser Leu Ser Trp Phe Gln Gln Lys Pro Asp Gly     50 55 60 Ser Val Lys Arg Leu Ile Tyr Ser Thr Ser Thr Leu Glu Ser Gly Val 65 70 75 80 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Ser                 85 90 95 Ile Thr Ser Leu Glu Ser Glu Asp Phe Ala Met Tyr Tyr Cys Leu Gln             100 105 110 Phe Ala Thr Tyr Pro Asp Thr Phe Gly Thr Gly Thr Lys Leu Glu Leu         115 120 125 Arg Arg Ala Asp Ala Ala Xaa Thr     130 135 <210> 16 <211> 471 <212> DNA <213> Rattus rattus <220> <221> misc_feature <222> (469). (469) <223> n is a, c, g, or t <400> 16 atggaatgga actgggtctt tctcttcctc ctgtcagtaa ctgcagaagt ccagtcccag 60 gtccagctgc agcagtctgg agcggagctg gcaaagcctg gctcctcaat gaagatttcc 120 tgcaaggctt ccggctaccc cgtcaccagc tattatataa gttggataaa gcagacgact 180 ggacagggcc ttgagtatat tggatatgtt gacatgggaa atggacggac taactacaat 240 gagaagttca agggcaaggc cacattgact gtagacaaat cctccagcac agccttcatg 300 caactcagca gcctgacacc tgacgactct gcggtctatt actgtgcaag ggactccaac 360 tggggggttg attactgggg ccaaggagtc atggtcacag tctcctcagc tgaaacaaca 420 gccccatctg tctatccact ggctcctgga actgctctca aaagtaacnc c 471 <210> 17 <211> 157 <212> PRT <213> Rattus rattus <220> <221> misc_feature &Lt; 222 > (157) <223> Xaa can be any naturally occurring amino acid <400> 17 Met Glu Trp Asn Trp Val Phe Leu Phe Leu Leu Ser Val Thr Ala Glu 1 5 10 15 Val Gln Ser Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Lys             20 25 30 Pro Gly Ser Ser Met Lys Ser Ser Cys Lys Ser Ser Gly Tyr Pro Val         35 40 45 Thr Ser Tyr Tyr Ile Ser Trp Ile Lys Gln Thr Thr Gly Gln Gly Leu     50 55 60 Glu Tyr Ile Gly Tyr Val Asp Met Gly Asn Gly Arg Thr Asn Tyr Asn 65 70 75 80 Glu Lys Phe Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser                 85 90 95 Thr Ala Phe Met Gln Leu Ser Ser Leu Thr Pro Asp Asp Ser Ala Val             100 105 110 Tyr Tyr Cys Ala Arg Asp Ser Asn Trp Gly Val Asp Tyr Trp Gly Gln         115 120 125 Gly Val Met Val Thr Val Ser Ser Ala Glu Thr Ala Pro Ser Val     130 135 140 Tyr Pro Leu Ala Pro Gly Thr Ala Leu Lys Ser Asn Xaa 145 150 155 <210> 18 <211> 449 <212> DNA <213> artificial <220> <223> Nucleotide Sequence coding Human Light Chain Secretory Signal and        Human Kappa Constant Region <400> 18 gcctccggac tctagagcca ccatggtgct gcagacccag gtgttcatct ccctgctgct 60 gtggatctcc ggcgcgtacg gcgatatcgt gatgattaaa cgtacggtgg ccgccccctc 120 cgtgttcatc ttccccccct ccgacgagca gctgaagtcc ggcaccgcct ccgtggtgtg 180 cctgctgaat aacttctacc ccagagaggc caaggtgcag tggaaggtgg acaacgccct 240 gcagtccggg aactcccagg agagcgtgac cgagcaggac agcaaggaca gcacctacag 300 cctgagcagc accctgaccc tgagcaaagc cgactacgag aagcacaagg tgtacgcctg 360 cgaggtgacc caccagggcc tgagctcccc cgtcaccaag agcttcaaca ggggggagtg 420 ttaggggccc gtttaaacgg gggaggcta 449 <210> 19 <211> 30 <212> DNA <213> artificial <220> <223> PCR Primer 3.3-F1 <400> 19 tataccgtcg acctctagct agagcttggc 30 <210> 20 <211> 30 <212> DNA <213> artificial <220> <223> PCR Primer 3.3-R1 <400> 20 gctatggcag ggcctgccgc cccgacgttg 30 <210> 21 <211> 1132 <212> DNA <213> artificial <220> <223> Nucleotide Sequence coding Human Heavy Chain Secretory Signal and        Human IgG1 Constant Region <400> 21 gcctccggac tctagagcca ccatgaaaca cctgtggttc ttcctcctgc tggtggcagc 60 tcccagatgg gtgctgagcc aggtgcaatt gtgcaggcgg ttagctcagc ctccaccaag 120 ggcccaagcg tcttccccct ggcaccctcc tccaagagca cctctggcgg cacagccgcc 180 ctgggctgcc tggtcaagga ctacttcccc gaacccgtga ccgtgagctg gaactcaggc 240 gccctgacca gcggcgtgca caccttcccc gctgtcctgc agtcctcagg actctactcc 300 ctcagcagcg tggtgaccgt gccctccagc agcttgggca cccagaccta catctgcaac 360 gtgaatcaca agcccagcaa caccaaggtg gacaagagag ttgagcccaa atcttgtgac 420 aaaactcaca catgcccacc ctgcccagca cctgaactcc tggggggacc ctcagtcttc 480 ctcttccccc caaaacccaa ggacaccctc atgatctccc ggacccctga ggtcacatgc 540 gtggtggtgg acgtgagcca cgaagaccct gaggtcaagt tcaactggta cgtggacggc 600 gtggaggtgc ataatgccaa gacaaagccc cgggaggagc agtacaacag cacgtaccgg 660 gtggtcagcg tcctcaccgt cctgcaccag gactggctta atggcaagga gtacaagtgc 720 aaggtctcca acaaagccct cccagccccc atcgagaaaa ccatctccaa agccaaaggc 780 cagccccggg aaccacaggt gtacaccctg cccccatccc gggaggagat gaccaagaac 840 caggtcagcc tgacctgcct ggtcaaaggc ttctatccca gcgacatcgc cgtggagtgg 900 gagagcaatg gccagcccga gaacaactac aagaccaccc ctcccgtgct ggactccgac 960 ggctccttct tcctctacag caagctcacc gtggacaaga gcaggtggca gcagggcaac 1020 gtcttctcat gctccgtgat gcatgaggct ctgcacaacc actacaccca gaagagcctc 1080 tccctgtctc ccggcaaatg agatatcggg cccgtttaaa cgggggaggc ta 1132 <210> 22 <211> 705 <212> DNA <213> artificial <220> <223> Nucleotide Sequence coding cR118_L <400> 22 atggtgctgc agacccaggt gttcatctcc ctgctgctgt ggatctccgg cgcgtacggc 60 gacatccagt tgacccagtc tccatccaca ttgcctgcat ccctgggaga gagagtcacc 120 atcagttgca gagcaagtca gagtattagc aatagtttaa gctggtttca acagaaacca 180 gatggaactg ttaaacgcct gatctattct acatccactt tagaatctgg tgtcccatca 240 aggttcagtg gcagtgggtc tgggacagat tattctctct ccatcaccag tcttgagtct 300 gaagattttg caatgtatta ctgtctacag tttgctactt ttccggacac gtttggaact 360 gggaccaaac tggaactgag acgggctgtg gccgccccct ccgtgttcat cttccccccc 420 tccgacgagc agctgaagtc cggcaccgcc tccgtggtgt gcctgctgaa taacttctac 480 cccagagagg ccaaggtgca gtggaaggtg gacaacgccc tgcagtccgg gaactcccag 540 gagagcgtga ccgagcagga cagcaaggac agcacctaca gcctgagcag caccctgacc 600 ctgagcaaag ccgactacga gaagcacaag gtgtacgcct gcgaggtgac ccaccagggc 660 ctgagctccc ccgtcaccaa gagcttcaac aggggggagt gttag 705 <210> 23 <211> 234 <212> PRT <213> artificial <220> <223> Amino Acid Sequence of cR118_L <400> 23 Met Val Leu Gln Thr Gln Val Phe Ile Ser Leu Leu Leu Trp Ile Ser 1 5 10 15 Gly Ala Tyr Gly Asp Ile Gln Leu Thr Gln Ser Pro Ser Thr Leu Pro             20 25 30 Ala Ser Leu Gly Glu Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Ser         35 40 45 Ile Ser Asn Ser Leu Ser Trp Phe Gln Gln Lys Pro Asp Gly Thr Val     50 55 60 Lys Arg Leu Ile Tyr Ser Thr Ser Thr Leu Glu Ser Gly Val Ser Ser 65 70 75 80 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Ser Ile Thr                 85 90 95 Ser Leu Glu Ser Glu Asp Phe Ala Met Tyr Tyr Cys Leu Gln Phe Ala             100 105 110 Thr Phe Pro Asp Thr Phe Gly Thr Gly Thr Lys Leu Glu Leu Arg Arg         115 120 125 Ala Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln     130 135 140 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 145 150 155 160 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser                 165 170 175 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr             180 185 190 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys         195 200 205 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro     210 215 220 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 <210> 24 <211> 705 <212> DNA <213> artificial <220> <223> Nucleotide Sequence coding cR198_L <400> 24 atggtgctgc agacccaggt gttcatctcc ctgctgctgt ggatctccgg cgcgtacggc 60 gacatccagt tgacccagtc tccatccaca ttgcctgcat ctctgggaga gagagtcacc 120 atcagttgca gagcaagtca gagtattggc aatagtttaa gctggtttca gcagaaacca 180 gatggatctg ttaaacgcct gatctactct acatccactt tagaatctgg tgtcccatca 240 aggttcagtg gcagtgggtc tgggacagat tattctctct ccatcaccag tcttgagtct 300 gaagattttg caatgtatta ctgtctacag tttgctactt atccggacac gtttggaact 360 gggaccaaac tggaactgag acgggctgtg gccgccccct ccgtgttcat cttccccccc 420 tccgacgagc agctgaagtc cggcaccgcc tccgtggtgt gcctgctgaa taacttctac 480 cccagagagg ccaaggtgca gtggaaggtg gacaacgccc tgcagtccgg gaactcccag 540 gagagcgtga ccgagcagga cagcaaggac agcacctaca gcctgagcag caccctgacc 600 ctgagcaaag ccgactacga gaagcacaag gtgtacgcct gcgaggtgac ccaccagggc 660 ctgagctccc ccgtcaccaa gagcttcaac aggggggagt gttag 705 <210> 25 <211> 234 <212> PRT <213> artificial <220> <223> Amino Acid Sequence of cR198_L <400> 25 Met Val Leu Gln Thr Gln Val Phe Ile Ser Leu Leu Leu Trp Ile Ser 1 5 10 15 Gly Ala Tyr Gly Asp Ile Gln Leu Thr Gln Ser Pro Ser Thr Leu Pro             20 25 30 Ala Ser Leu Gly Glu Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Ser         35 40 45 Ile Gly Asn Ser Leu Ser Trp Phe Gln Gln Lys Pro Asp Gly Ser Val     50 55 60 Lys Arg Leu Ile Tyr Ser Thr Ser Thr Leu Glu Ser Gly Val Ser Ser 65 70 75 80 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Ser Ile Thr                 85 90 95 Ser Leu Glu Ser Glu Asp Phe Ala Met Tyr Tyr Cys Leu Gln Phe Ala             100 105 110 Thr Tyr Pro Asp Thr Phe Gly Thr Gly Thr Lys Leu Glu Leu Arg Arg         115 120 125 Ala Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln     130 135 140 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 145 150 155 160 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser                 165 170 175 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr             180 185 190 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys         195 200 205 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro     210 215 220 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 <210> 26 <211> 1401 <212> DNA <213> artificial <220> <223> Nucleotide Sequence coding cR118_H <400> 26 atgaaacacc tgtggttctt cctcctgctg gtggcagctc ccagatgggt gctgagccag 60 gtccagctgc agcagtctgg agcggagctg gcaaagcctg gctcctcagt gaagatttcc 120 tgcaaggctt ccggctacac cgtcaccgcc tattatataa gttggataag gcagacgatt 180 ggacagggcc ttgagtatgt tggatatatt gacatgggaa atggaaggac taactacaat 240 gcgaggttca agggcaaggc cacattgact gtggacaaat cctccagcac agccttcatg 300 caactcagca gcctgacacc tgacgactct gcggtctatt actgtgcaag ggactccaac 360 tggggggttg attactgggg ccaaggagtc atggtcacag tcagctcagc ctccaccaag 420 ggcccaagcg tcttccccct ggcaccctcc tccaagagca cctctggcgg cacagccgcc 480 ctgggctgcc tggtcaagga ctacttcccc gaacccgtga ccgtgagctg gaactcaggc 540 gccctgacca gcggcgtgca caccttcccc gctgtcctgc agtcctcagg actctactcc 600 ctcagcagcg tggtgaccgt gccctccagc agcttgggca cccagaccta catctgcaac 660 gtgaatcaca agcccagcaa caccaaggtg gacaagagag ttgagcccaa atcttgtgac 720 aaaactcaca catgcccacc ctgcccagca cctgaactcc tggggggacc ctcagtcttc 780 ctcttccccc caaaacccaa ggacaccctc atgatctccc ggacccctga ggtcacatgc 840 gtggtggtgg acgtgagcca cgaagaccct gaggtcaagt tcaactggta cgtggacggc 900 gtggaggtgc ataatgccaa gacaaagccc cgggaggagc agtacaacag cacgtaccgg 960 gtggtcagcg tcctcaccgt cctgcaccag gactggctta atggcaagga gtacaagtgc 1020 aaggtctcca acaaagccct cccagccccc atcgagaaaa ccatctccaa agccaaaggc 1080 cagccccggg aaccacaggt gtacaccctg cccccatccc gggaggagat gaccaagaac 1140 caggtcagcc tgacctgcct ggtcaaaggc ttctatccca gcgacatcgc cgtggagtgg 1200 gagagcaatg gccagcccga gaacaactac aagaccaccc ctcccgtgct ggactccgac 1260 ggctccttct tcctctacag caagctcacc gtggacaaga gcaggtggca gcagggcaac 1320 gtcttctcat gctccgtgat gcatgaggct ctgcacaacc actacaccca gaagagcctc 1380 tccctgtctc ccggcaaatg a 1401 <210> 27 <211> 466 <212> PRT <213> artificial <220> <223> Amino Acid Sequence of cR118_H <400> 27 Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala Pro Arg Trp 1 5 10 15 Val Leu Ser Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Lys             20 25 30 Pro Gly Ser Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Val         35 40 45 Thr Ala Tyr Ile Ser Trp Ile Arg Gln Thr Ile Gly Gln Gly Leu     50 55 60 Glu Tyr Val Gly Tyr Ile Asp Met Gly Asn Gly Arg Thr Asn Tyr Asn 65 70 75 80 Ala Arg Phe Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser                 85 90 95 Thr Ala Phe Met Gln Leu Ser Ser Leu Thr Pro Asp Asp Ser Ala Val             100 105 110 Tyr Tyr Cys Ala Arg Asp Ser Asn Trp Gly Val Asp Tyr Trp Gly Gln         115 120 125 Gly Val Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val     130 135 140 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 145 150 155 160 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser                 165 170 175 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val             180 185 190 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro         195 200 205 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys     210 215 220 Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp 225 230 235 240 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly                 245 250 255 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile             260 265 270 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu         275 280 285 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His     290 295 300 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 305 310 315 320 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys                 325 330 335 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu             340 345 350 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr         355 360 365 Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu     370 375 380 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 385 390 395 400 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val                 405 410 415 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp             420 425 430 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His         435 440 445 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro     450 455 460 Gly Lys 465 <210> 28 <211> 1401 <212> DNA <213> artificial <220> <223> Nucleotide Sequence coding cR198_H <400> 28 atgaaacacc tgtggttctt cctcctgctg gtggcagctc ccagatgggt gctgagccag 60 gtccagctgc agcagtctgg agcggagctg gcaaagcctg gctcctcaat gaagatttcc 120 tgcaaggctt ccggctaccc cgtcaccagc tattatataa gttggataaa gcagacgact 180 ggacagggcc ttgagtatat tggatatgtt gacatgggaa atggacggac taactacaat 240 gagaagttca agggcaaggc cacattgact gtagacaaat cctccagcac agccttcatg 300 caactcagca gcctgacacc tgacgactct gcggtctatt actgtgcaag ggactccaac 360 tggggggttg attactgggg ccaaggagtc atggtcacag tcagctcagc ctccaccaag 420 ggcccaagcg tcttccccct ggcaccctcc tccaagagca cctctggcgg cacagccgcc 480 ctgggctgcc tggtcaagga ctacttcccc gaacccgtga ccgtgagctg gaactcaggc 540 gccctgacca gcggcgtgca caccttcccc gctgtcctgc agtcctcagg actctactcc 600 ctcagcagcg tggtgaccgt gccctccagc agcttgggca cccagaccta catctgcaac 660 gtgaatcaca agcccagcaa caccaaggtg gacaagagag ttgagcccaa atcttgtgac 720 aaaactcaca catgcccacc ctgcccagca cctgaactcc tggggggacc ctcagtcttc 780 ctcttccccc caaaacccaa ggacaccctc atgatctccc ggacccctga ggtcacatgc 840 gtggtggtgg acgtgagcca cgaagaccct gaggtcaagt tcaactggta cgtggacggc 900 gtggaggtgc ataatgccaa gacaaagccc cgggaggagc agtacaacag cacgtaccgg 960 gtggtcagcg tcctcaccgt cctgcaccag gactggctta atggcaagga gtacaagtgc 1020 aaggtctcca acaaagccct cccagccccc atcgagaaaa ccatctccaa agccaaaggc 1080 cagccccggg aaccacaggt gtacaccctg cccccatccc gggaggagat gaccaagaac 1140 caggtcagcc tgacctgcct ggtcaaaggc ttctatccca gcgacatcgc cgtggagtgg 1200 gagagcaatg gccagcccga gaacaactac aagaccaccc ctcccgtgct ggactccgac 1260 ggctccttct tcctctacag caagctcacc gtggacaaga gcaggtggca gcagggcaac 1320 gtcttctcat gctccgtgat gcatgaggct ctgcacaacc actacaccca gaagagcctc 1380 tccctgtctc ccggcaaatg a 1401 <210> 29 <211> 466 <212> PRT <213> artificial <220> <223> Amino Acid Sequence of cR198_H <400> 29 Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala Pro Arg Trp 1 5 10 15 Val Leu Ser Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Lys             20 25 30 Pro Gly Ser Ser Met Lys Ser Ser Cys Lys Ser Ser Gly Tyr Pro Val         35 40 45 Thr Ser Tyr Tyr Ile Ser Trp Ile Lys Gln Thr Thr Gly Gln Gly Leu     50 55 60 Glu Tyr Ile Gly Tyr Val Asp Met Gly Asn Gly Arg Thr Asn Tyr Asn 65 70 75 80 Glu Lys Phe Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser                 85 90 95 Thr Ala Phe Met Gln Leu Ser Ser Leu Thr Pro Asp Asp Ser Ala Val             100 105 110 Tyr Tyr Cys Ala Arg Asp Ser Asn Trp Gly Val Asp Tyr Trp Gly Gln         115 120 125 Gly Val Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val     130 135 140 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 145 150 155 160 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser                 165 170 175 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val             180 185 190 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro         195 200 205 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys     210 215 220 Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp 225 230 235 240 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly                 245 250 255 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile             260 265 270 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu         275 280 285 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His     290 295 300 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 305 310 315 320 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys                 325 330 335 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu             340 345 350 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr         355 360 365 Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu     370 375 380 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 385 390 395 400 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val                 405 410 415 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp             420 425 430 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His         435 440 445 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro     450 455 460 Gly Lys 465 <210> 30 <211> 705 <212> DNA <213> artificial <220> <223> Nucleotide Sequence coding hR198_L1 <400> 30 atggtgctgc agacccaggt gttcatctcc ctgctgctgt ggatctccgg cgcgtacggc 60 gatatccagc tgacccagag ccctagcagc ctgtctgcca gcgtgggcga cagagtgacc 120 atcacctgta gagccagcca gagcatcggc aacagcctga gctggttcca gcagaaaccc 180 ggcaaggccc ccaagcggct gatctacagc accagcaccc tggaaagcgg cgtgcccagc 240 agattttctg gcagcggctc cggcaccgac tacaccctga caatcagcag cctgcagccc 300 gaggacttcg ccatgtacta ctgcctgcag ttcgccacct accccgacac ctttggccag 360 ggcaccaagg tggaaatcaa gcgtacggtg gccgccccct ccgtgttcat cttccccccc 420 tccgacgagc agctgaagtc cggcaccgcc tccgtggtgt gcctgctgaa taacttctac 480 cccagagagg ccaaggtgca gtggaaggtg gacaacgccc tgcagtccgg gaactcccag 540 gagagcgtga ccgagcagga cagcaaggac agcacctaca gcctgagcag caccctgacc 600 ctgagcaaag ccgactacga gaagcacaag gtgtacgcct gcgaggtgac ccaccagggc 660 ctgagctccc ccgtcaccaa gagcttcaac aggggggagt gttag 705 <210> 31 <211> 234 <212> PRT <213> artificial <220> <223> Amino Acid Sequence of hR198_L1 <400> 31 Met Val Leu Gln Thr Gln Val Phe Ile Ser Leu Leu Leu Trp Ile Ser 1 5 10 15 Gly Ala Tyr Gly Asp Ile Gln Leu Thr Gln Ser Ser Ser Ser Leu Ser             20 25 30 Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser         35 40 45 Ile Gly Asn Ser Leu Ser Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro     50 55 60 Lys Arg Leu Ile Tyr Ser Thr Ser Thr Leu Glu Ser Gly Val Ser Ser 65 70 75 80 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser                 85 90 95 Ser Leu Gln Pro Glu Asp Phe Ala Met Tyr Tyr Cys Leu Gln Phe Ala             100 105 110 Thr Tyr Pro Asp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg         115 120 125 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln     130 135 140 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 145 150 155 160 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser                 165 170 175 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr             180 185 190 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys         195 200 205 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro     210 215 220 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 <210> 32 <211> 705 <212> DNA <213> artificial <220> <223> Nucleotide Sequence coding hR198_L2 <400> 32 atggtgctgc agacccaggt gttcatctcc ctgctgctgt ggatctccgg cgcgtacggc 60 gatatccagc tgacccagag ccctagcagc ctgtctgcca gcgtgggcga cagagtgacc 120 atcacctgta gagccagcca gagcatcggc aacagcctga gctggttcca gcagaaaccc 180 ggcaaggccg tgaagcggct gatctacagc accagcaccc tggaaagcgg cgtgcccagc 240 agattttctg gcagcggctc cggcaccgac tacaccctga caatcagcag cctgcagccc 300 gaggacttcg ccatgtacta ctgcctgcag ttcgccacct accccgacac ctttggccag 360 ggcaccaagg tggaaatcaa gcgtacggtg gccgccccct ccgtgttcat cttccccccc 420 tccgacgagc agctgaagtc cggcaccgcc tccgtggtgt gcctgctgaa taacttctac 480 cccagagagg ccaaggtgca gtggaaggtg gacaacgccc tgcagtccgg gaactcccag 540 gagagcgtga ccgagcagga cagcaaggac agcacctaca gcctgagcag caccctgacc 600 ctgagcaaag ccgactacga gaagcacaag gtgtacgcct gcgaggtgac ccaccagggc 660 ctgagctccc ccgtcaccaa gagcttcaac aggggggagt gttag 705 <210> 33 <211> 234 <212> PRT <213> artificial <220> <223> Amino Acid Sequence of hR198_L2 <400> 33 Met Val Leu Gln Thr Gln Val Phe Ile Ser Leu Leu Leu Trp Ile Ser 1 5 10 15 Gly Ala Tyr Gly Asp Ile Gln Leu Thr Gln Ser Ser Ser Ser Leu Ser             20 25 30 Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser         35 40 45 Ile Gly Asn Ser Leu Ser Trp Phe Gln Gln Lys Pro Gly Lys Ala Val     50 55 60 Lys Arg Leu Ile Tyr Ser Thr Ser Thr Leu Glu Ser Gly Val Ser Ser 65 70 75 80 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser                 85 90 95 Ser Leu Gln Pro Glu Asp Phe Ala Met Tyr Tyr Cys Leu Gln Phe Ala             100 105 110 Thr Tyr Pro Asp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg         115 120 125 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln     130 135 140 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 145 150 155 160 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser                 165 170 175 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr             180 185 190 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys         195 200 205 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro     210 215 220 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 <210> 34 <211> 705 <212> DNA <213> artificial <220> <223> Nucleotide Sequence coding hR198_L3 <400> 34 atggtgctgc agacccaggt gttcatctcc ctgctgctgt ggatctccgg cgcgtacggc 60 gatatccagc tgacccagag ccctagcagc ctgtctgcca gcgtgggcga cagagtgacc 120 atcacctgta gagccagcca gagcatcggc aacagcctga gctggttcca gcagaaaccc 180 ggcaaggccc ccaagcggct gatctacagc accagcaccc tggaaagcgg cgtgcccagc 240 agattttctg gcagcggctc cggcaccgac tacaccctga caatcagcag cctgcagccc 300 gaggacttcg ccatgtacta ctgcctgcag ttcgccacct tccccgacac ctttggccag 360 ggcaccaagg tggaaatcaa gcgtacggtg gccgccccct ccgtgttcat cttccccccc 420 tccgacgagc agctgaagtc cggcaccgcc tccgtggtgt gcctgctgaa taacttctac 480 cccagagagg ccaaggtgca gtggaaggtg gacaacgccc tgcagtccgg gaactcccag 540 gagagcgtga ccgagcagga cagcaaggac agcacctaca gcctgagcag caccctgacc 600 ctgagcaaag ccgactacga gaagcacaag gtgtacgcct gcgaggtgac ccaccagggc 660 ctgagctccc ccgtcaccaa gagcttcaac aggggggagt gttag 705 <210> 35 <211> 234 <212> PRT <213> artificial <220> <223> Amino Acid Sequence of hR198_L3 <400> 35 Met Val Leu Gln Thr Gln Val Phe Ile Ser Leu Leu Leu Trp Ile Ser 1 5 10 15 Gly Ala Tyr Gly Asp Ile Gln Leu Thr Gln Ser Ser Ser Ser Leu Ser             20 25 30 Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser         35 40 45 Ile Gly Asn Ser Leu Ser Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro     50 55 60 Lys Arg Leu Ile Tyr Ser Thr Ser Thr Leu Glu Ser Gly Val Ser Ser 65 70 75 80 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser                 85 90 95 Ser Leu Gln Pro Glu Asp Phe Ala Met Tyr Tyr Cys Leu Gln Phe Ala             100 105 110 Thr Phe Pro Asp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg         115 120 125 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln     130 135 140 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 145 150 155 160 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser                 165 170 175 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr             180 185 190 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys         195 200 205 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro     210 215 220 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 <210> 36 <211> 705 <212> DNA <213> artificial <220> <223> Nucleotide Sequence coding hR198_L4 <400> 36 atggtgctgc agacccaggt gttcatctcc ctgctgctgt ggatctccgg cgcgtacggc 60 gatatccagc tgacccagag ccctagcagc ctgtctgcca gcgtgggcga cagagtgacc 120 atcacctgta gagccagcca gagcatcggc aacagcctga gctggttcca gcagaaaccc 180 ggcaaggccg tgaagcggct gatctacagc accagcaccc tggaaagcgg cgtgcccagc 240 agattttctg gcagcggctc cggcaccgac tacaccctga caatcagcag cctgcagccc 300 gaggacttcg ccatgtacta ctgcctgcag ttcgccacct tccccgacac ctttggccag 360 ggcaccaagg tggaaatcaa gcgtacggtg gccgccccct ccgtgttcat cttccccccc 420 tccgacgagc agctgaagtc cggcaccgcc tccgtggtgt gcctgctgaa taacttctac 480 cccagagagg ccaaggtgca gtggaaggtg gacaacgccc tgcagtccgg gaactcccag 540 gagagcgtga ccgagcagga cagcaaggac agcacctaca gcctgagcag caccctgacc 600 ctgagcaaag ccgactacga gaagcacaag gtgtacgcct gcgaggtgac ccaccagggc 660 ctgagctccc ccgtcaccaa gagcttcaac aggggggagt gttag 705 <210> 37 <211> 234 <212> PRT <213> artificial <220> <223> Amino Acid Sequence of hR198_L4 <400> 37 Met Val Leu Gln Thr Gln Val Phe Ile Ser Leu Leu Leu Trp Ile Ser 1 5 10 15 Gly Ala Tyr Gly Asp Ile Gln Leu Thr Gln Ser Ser Ser Ser Leu Ser             20 25 30 Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser         35 40 45 Ile Gly Asn Ser Leu Ser Trp Phe Gln Gln Lys Pro Gly Lys Ala Val     50 55 60 Lys Arg Leu Ile Tyr Ser Thr Ser Thr Leu Glu Ser Gly Val Ser Ser 65 70 75 80 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser                 85 90 95 Ser Leu Gln Pro Glu Asp Phe Ala Met Tyr Tyr Cys Leu Gln Phe Ala             100 105 110 Thr Phe Pro Asp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg         115 120 125 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln     130 135 140 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 145 150 155 160 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser                 165 170 175 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr             180 185 190 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys         195 200 205 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro     210 215 220 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 <210> 38 <211> 1401 <212> DNA <213> artificial <220> <223> Nucleotide Sequence coding hR198_H1 <400> 38 atgaaacacc tgtggttctt cctcctgctg gtggcagctc ccagatgggt gctgagccag 60 gtgcagctgg tgcagtctgg cgccgaagtg aagaaaccag gcgccagcgt gaaggtgtcc 120 tgcaaggcct ctggctaccc cgtgaccagc tactacatca gctgggtcag acaggcccca 180 ggccagggcc tggaatacat cggctatgtg gacatgggca acggccggac caactacaac 240 gagaagttca agggcagagc caccctgacc gtggacaaga gcaccagcac cgcctacatg 300 gaactgagca gcctgcggag cgaggacacc gccgtgtact actgcgccag agacagcaac 360 tggggcgtgg actattgggg ccagggcaca ctcgtgaccg tcagctcagc ctccaccaag 420 ggcccaagcg tcttccccct ggcaccctcc tccaagagca cctctggcgg cacagccgcc 480 ctgggctgcc tggtcaagga ctacttcccc gaacccgtga ccgtgagctg gaactcaggc 540 gccctgacca gcggcgtgca caccttcccc gctgtcctgc agtcctcagg actctactcc 600 ctcagcagcg tggtgaccgt gccctccagc agcttgggca cccagaccta catctgcaac 660 gtgaatcaca agcccagcaa caccaaggtg gacaagagag ttgagcccaa atcttgtgac 720 aaaactcaca catgcccacc ctgcccagca cctgaactcc tggggggacc ctcagtcttc 780 ctcttccccc caaaacccaa ggacaccctc atgatctccc ggacccctga ggtcacatgc 840 gtggtggtgg acgtgagcca cgaagaccct gaggtcaagt tcaactggta cgtggacggc 900 gtggaggtgc ataatgccaa gacaaagccc cgggaggagc agtacaacag cacgtaccgg 960 gtggtcagcg tcctcaccgt cctgcaccag gactggctta atggcaagga gtacaagtgc 1020 aaggtctcca acaaagccct cccagccccc atcgagaaaa ccatctccaa agccaaaggc 1080 cagccccggg aaccacaggt gtacaccctg cccccatccc gggaggagat gaccaagaac 1140 caggtcagcc tgacctgcct ggtcaaaggc ttctatccca gcgacatcgc cgtggagtgg 1200 gagagcaatg gccagcccga gaacaactac aagaccaccc ctcccgtgct ggactccgac 1260 ggctccttct tcctctacag caagctcacc gtggacaaga gcaggtggca gcagggcaac 1320 gtcttctcat gctccgtgat gcatgaggct ctgcacaacc actacaccca gaagagcctc 1380 tccctgtctc ccggcaaatg a 1401 <210> 39 <211> 466 <212> PRT <213> artificial <220> <223> Amino Acid Sequence of hR198_H1 <400> 39 Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala Pro Arg Trp 1 5 10 15 Val Leu Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys             20 25 30 Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Pro Val         35 40 45 Thr Ser Tyr Tyr Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu     50 55 60 Glu Tyr Ile Gly Tyr Val Asp Met Gly Asn Gly Arg Thr Asn Tyr Asn 65 70 75 80 Glu Lys Phe Lys Gly Arg Ala Thr Leu Thr Val Asp Lys Ser Thr Ser                 85 90 95 Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val             100 105 110 Tyr Tyr Cys Ala Arg Asp Ser Asn Trp Gly Val Asp Tyr Trp Gly Gln         115 120 125 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Ser Ser Val     130 135 140 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 145 150 155 160 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser                 165 170 175 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val             180 185 190 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro         195 200 205 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys     210 215 220 Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp 225 230 235 240 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly                 245 250 255 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile             260 265 270 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu         275 280 285 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His     290 295 300 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 305 310 315 320 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys                 325 330 335 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu             340 345 350 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr         355 360 365 Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu     370 375 380 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 385 390 395 400 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val                 405 410 415 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp             420 425 430 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His         435 440 445 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro     450 455 460 Gly Lys 465 <210> 40 <211> 1401 <212> DNA <213> artificial <220> <223> Nucleotide Sequence coding hR198_H2 <400> 40 atgaaacacc tgtggttctt cctcctgctg gtggcagctc ccagatgggt gctgagccag 60 gtgcagctgg tgcagtctgg cgccgaagtg aagaaaccag gcgccagcgt gaaggtgtcc 120 tgcaaggcct ctggctaccc cgtgaccagc tactacatca gctggatcag acaggcccca 180 ggccagggcc tggaatacat cggctatgtg gacatgggca acggccggac caactacaac 240 gagaagttca agggcagagc caccctgacc gtggacaaga gcaccagcac cgcctacatg 300 gaactgagca gcctgcggag cgaggacacc gccgtgtact actgcgccag agacagcaac 360 tggggcgtgg actattgggg ccagggcaca ctcgtgaccg tcagctcagc ctccaccaag 420 ggcccaagcg tcttccccct ggcaccctcc tccaagagca cctctggcgg cacagccgcc 480 ctgggctgcc tggtcaagga ctacttcccc gaacccgtga ccgtgagctg gaactcaggc 540 gccctgacca gcggcgtgca caccttcccc gctgtcctgc agtcctcagg actctactcc 600 ctcagcagcg tggtgaccgt gccctccagc agcttgggca cccagaccta catctgcaac 660 gtgaatcaca agcccagcaa caccaaggtg gacaagagag ttgagcccaa atcttgtgac 720 aaaactcaca catgcccacc ctgcccagca cctgaactcc tggggggacc ctcagtcttc 780 ctcttccccc caaaacccaa ggacaccctc atgatctccc ggacccctga ggtcacatgc 840 gtggtggtgg acgtgagcca cgaagaccct gaggtcaagt tcaactggta cgtggacggc 900 gtggaggtgc ataatgccaa gacaaagccc cgggaggagc agtacaacag cacgtaccgg 960 gtggtcagcg tcctcaccgt cctgcaccag gactggctta atggcaagga gtacaagtgc 1020 aaggtctcca acaaagccct cccagccccc atcgagaaaa ccatctccaa agccaaaggc 1080 cagccccggg aaccacaggt gtacaccctg cccccatccc gggaggagat gaccaagaac 1140 caggtcagcc tgacctgcct ggtcaaaggc ttctatccca gcgacatcgc cgtggagtgg 1200 gagagcaatg gccagcccga gaacaactac aagaccaccc ctcccgtgct ggactccgac 1260 ggctccttct tcctctacag caagctcacc gtggacaaga gcaggtggca gcagggcaac 1320 gtcttctcat gctccgtgat gcatgaggct ctgcacaacc actacaccca gaagagcctc 1380 tccctgtctc ccggcaaatg a 1401 <210> 41 <211> 466 <212> PRT <213> artificial <220> <223> Amino Acid Sequence of hR198_H2 <400> 41 Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala Pro Arg Trp 1 5 10 15 Val Leu Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys             20 25 30 Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Pro Val         35 40 45 Thr Ser Tyr Tyr Ile Ser Trp Ile Arg Gln Ala Pro Gly Gln Gly Leu     50 55 60 Glu Tyr Ile Gly Tyr Val Asp Met Gly Asn Gly Arg Thr Asn Tyr Asn 65 70 75 80 Glu Lys Phe Lys Gly Arg Ala Thr Leu Thr Val Asp Lys Ser Thr Ser                 85 90 95 Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val             100 105 110 Tyr Tyr Cys Ala Arg Asp Ser Asn Trp Gly Val Asp Tyr Trp Gly Gln         115 120 125 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Ser Ser Val     130 135 140 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 145 150 155 160 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser                 165 170 175 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val             180 185 190 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro         195 200 205 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys     210 215 220 Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp 225 230 235 240 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly                 245 250 255 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile             260 265 270 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu         275 280 285 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His     290 295 300 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 305 310 315 320 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys                 325 330 335 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu             340 345 350 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr         355 360 365 Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu     370 375 380 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 385 390 395 400 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val                 405 410 415 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp             420 425 430 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His         435 440 445 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro     450 455 460 Gly Lys 465 <210> 42 <211> 1401 <212> DNA <213> artificial <220> <223> Nucleotide Sequence coding hR198_H3 <400> 42 atgaaacacc tgtggttctt cctcctgctg gtggcagctc ccagatgggt gctgagccag 60 gtgcagctgg tgcagtctgg cgccgaagtg aagaaaccag gcgccagcgt gaaggtgtcc 120 tgcaaggcct ctggctaccc cgtgaccgcc tactacatca gctgggtcag acaggcccca 180 ggccagggcc tggaatacgt gggctacatc gacatgggca acggccggac caactacaac 240 gcccggttta agggcagagc caccctgacc gtggacaaga gcaccagcac cgcctacatg 300 gaactgagca gcctgcggag cgaggacacc gccgtgtact actgcgccag agacagcaac 360 tggggcgtgg actattgggg ccagggcaca ctcgtgaccg tcagctcagc ctccaccaag 420 ggcccaagcg tcttccccct ggcaccctcc tccaagagca cctctggcgg cacagccgcc 480 ctgggctgcc tggtcaagga ctacttcccc gaacccgtga ccgtgagctg gaactcaggc 540 gccctgacca gcggcgtgca caccttcccc gctgtcctgc agtcctcagg actctactcc 600 ctcagcagcg tggtgaccgt gccctccagc agcttgggca cccagaccta catctgcaac 660 gtgaatcaca agcccagcaa caccaaggtg gacaagagag ttgagcccaa atcttgtgac 720 aaaactcaca catgcccacc ctgcccagca cctgaactcc tggggggacc ctcagtcttc 780 ctcttccccc caaaacccaa ggacaccctc atgatctccc ggacccctga ggtcacatgc 840 gtggtggtgg acgtgagcca cgaagaccct gaggtcaagt tcaactggta cgtggacggc 900 gtggaggtgc ataatgccaa gacaaagccc cgggaggagc agtacaacag cacgtaccgg 960 gtggtcagcg tcctcaccgt cctgcaccag gactggctta atggcaagga gtacaagtgc 1020 aaggtctcca acaaagccct cccagccccc atcgagaaaa ccatctccaa agccaaaggc 1080 cagccccggg aaccacaggt gtacaccctg cccccatccc gggaggagat gaccaagaac 1140 caggtcagcc tgacctgcct ggtcaaaggc ttctatccca gcgacatcgc cgtggagtgg 1200 gagagcaatg gccagcccga gaacaactac aagaccaccc ctcccgtgct ggactccgac 1260 ggctccttct tcctctacag caagctcacc gtggacaaga gcaggtggca gcagggcaac 1320 gtcttctcat gctccgtgat gcatgaggct ctgcacaacc actacaccca gaagagcctc 1380 tccctgtctc ccggcaaatg a 1401 <210> 43 <211> 466 <212> PRT <213> artificial <220> <223> Amino Acid Sequence of hR198_H3 <400> 43 Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala Pro Arg Trp 1 5 10 15 Val Leu Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys             20 25 30 Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Pro Val         35 40 45 Thr Ala Tyr Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu     50 55 60 Glu Tyr Val Gly Tyr Ile Asp Met Gly Asn Gly Arg Thr Asn Tyr Asn 65 70 75 80 Ala Arg Phe Lys Gly Arg Ala Thr Leu Thr Val Asp Lys Ser Thr Ser                 85 90 95 Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val             100 105 110 Tyr Tyr Cys Ala Arg Asp Ser Asn Trp Gly Val Asp Tyr Trp Gly Gln         115 120 125 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Ser Ser Val     130 135 140 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 145 150 155 160 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser                 165 170 175 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val             180 185 190 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro         195 200 205 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys     210 215 220 Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp 225 230 235 240 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly                 245 250 255 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile             260 265 270 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu         275 280 285 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His     290 295 300 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 305 310 315 320 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys                 325 330 335 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu             340 345 350 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr         355 360 365 Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu     370 375 380 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 385 390 395 400 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val                 405 410 415 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp             420 425 430 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His         435 440 445 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro     450 455 460 Gly Lys 465 <210> 44 <211> 1401 <212> DNA <213> artificial <220> <223> Nucleotide Sequence coding hR198_H4 <400> 44 atgaaacacc tgtggttctt cctcctgctg gtggcagctc ccagatgggt gctgagccag 60 gtgcagctgg tgcagtctgg cgccgaagtg aagaaaccag gcgccagcgt gaaggtgtcc 120 tgcaaggcct ctggctaccc cgtgaccgcc tactacatca gctggatcag acaggcccca 180 ggccagggcc tggaatacgt gggctacatc gacatgggca acggccggac caactacaac 240 gcccggttta agggcagagc caccctgacc gtggacaaga gcaccagcac cgcctacatg 300 gaactgagca gcctgcggag cgaggacacc gccgtgtact actgcgccag agacagcaac 360 tggggcgtgg actattgggg ccagggcaca ctcgtgaccg tcagctcagc ctccaccaag 420 ggcccaagcg tcttccccct ggcaccctcc tccaagagca cctctggcgg cacagccgcc 480 ctgggctgcc tggtcaagga ctacttcccc gaacccgtga ccgtgagctg gaactcaggc 540 gccctgacca gcggcgtgca caccttcccc gctgtcctgc agtcctcagg actctactcc 600 ctcagcagcg tggtgaccgt gccctccagc agcttgggca cccagaccta catctgcaac 660 gtgaatcaca agcccagcaa caccaaggtg gacaagagag ttgagcccaa atcttgtgac 720 aaaactcaca catgcccacc ctgcccagca cctgaactcc tggggggacc ctcagtcttc 780 ctcttccccc caaaacccaa ggacaccctc atgatctccc ggacccctga ggtcacatgc 840 gtggtggtgg acgtgagcca cgaagaccct gaggtcaagt tcaactggta cgtggacggc 900 gtggaggtgc ataatgccaa gacaaagccc cgggaggagc agtacaacag cacgtaccgg 960 gtggtcagcg tcctcaccgt cctgcaccag gactggctta atggcaagga gtacaagtgc 1020 aaggtctcca acaaagccct cccagccccc atcgagaaaa ccatctccaa agccaaaggc 1080 cagccccggg aaccacaggt gtacaccctg cccccatccc gggaggagat gaccaagaac 1140 caggtcagcc tgacctgcct ggtcaaaggc ttctatccca gcgacatcgc cgtggagtgg 1200 gagagcaatg gccagcccga gaacaactac aagaccaccc ctcccgtgct ggactccgac 1260 ggctccttct tcctctacag caagctcacc gtggacaaga gcaggtggca gcagggcaac 1320 gtcttctcat gctccgtgat gcatgaggct ctgcacaacc actacaccca gaagagcctc 1380 tccctgtctc ccggcaaatg a 1401 <210> 45 <211> 466 <212> PRT <213> artificial <220> <223> Amino Acid Sequence of hR198_H4 <400> 45 Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala Pro Arg Trp 1 5 10 15 Val Leu Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys             20 25 30 Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Pro Val         35 40 45 Thr Ala Tyr Ile Ser Trp Ile Arg Gln Ala Pro Gly Gln Gly Leu     50 55 60 Glu Tyr Val Gly Tyr Ile Asp Met Gly Asn Gly Arg Thr Asn Tyr Asn 65 70 75 80 Ala Arg Phe Lys Gly Arg Ala Thr Leu Thr Val Asp Lys Ser Thr Ser                 85 90 95 Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val             100 105 110 Tyr Tyr Cys Ala Arg Asp Ser Asn Trp Gly Val Asp Tyr Trp Gly Gln         115 120 125 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Ser Ser Val     130 135 140 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 145 150 155 160 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser                 165 170 175 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val             180 185 190 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro         195 200 205 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys     210 215 220 Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp 225 230 235 240 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly                 245 250 255 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile             260 265 270 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu         275 280 285 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His     290 295 300 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 305 310 315 320 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys                 325 330 335 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu             340 345 350 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr         355 360 365 Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu     370 375 380 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 385 390 395 400 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val                 405 410 415 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp             420 425 430 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His         435 440 445 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro     450 455 460 Gly Lys 465 <210> 46 <211> 23 <212> DNA <213> artificial <220> <223> PCR Primer Orai1 HF <400> 46 atgcaagtcc aactggttca atc 23 <210> 47 <211> 21 <212> DNA <213> artificial <220> <223> PCR Primer Orai1 CH FR <400> 47 tgacggagcc agcgggaaga c 21 <210> 48 <211> 21 <212> DNA <213> artificial <220> <223> PCR Primer M13 rev long <400> 48 caggaaacag ctatgaccat g 21 <210> 49 <211> 31 <212> DNA <213> artificial <220> <223> PCR Primer SecM Stop R <400> 49 ctcgagttat tcattaggtg aggcgttgag g 31 <210> 50 <211> 24 <212> DNA <213> artificial <220> <223> PCR Primer Orai1 Lc F <400> 50 atggacattc aactgaccca aagc 24 <210> 51 <211> 24 <212> DNA <213> artificial <220> <223> PCR Primer Orai1 CL-FR <400> 51 gataaaaaca ctcggggccg ccac 24 <210> 52 <211> 63 <212> DNA <213> artificial <220> <223> PCR Primer Orai1 HR-FLAG R <400> 52 tcattatttg tcatcgtcat ctttatagtc gaattcttcg ccacgattaa aggatttggt 60 gac 63 <210> 53 <211> 63 <212> DNA <213> artificial <220> <223> PCR Primer Orai1 CL -FLAG R <400> 53 tcattatttg tcatcgtcat ctttatagtc gaattcttcg ccacgattaa aggatttggt 60 gac 63 <210> 54 <211> 30 <212> DNA <213> artificial <220> <223> PCR Primer Myc-R <400> 54 cagatcctcc tcagagatca gcttctgctc 30 <210> 55 <211> 705 <212> DNA <213> artificial <220> <223> Nucleotide Sequence coding hR198_LG1 <400> 55 atggtgctgc agacccaggt gttcatctcc ctgctgctgt ggatctccgg cgcgtacggc 60 gatatccagc tgacccagag ccctagcagc ctgtctgcca gcgtgggcga cagagtgacc 120 atcacctgta gagccagcca gagcatcggc ggcagcctga gctggttcca gcagaaaccc 180 ggcaaggccc ccaagcggct gatctacagc accagcaccc tggaaagcgg cgtgcccagc 240 agattttctg gcagcggctc cggcaccgac tacaccctga caatcagcag cctgcagccc 300 gaggacttcg ccatgtacta ctgcctgcag ttcgccatct tccccgacag ctttggccag 360 ggcaccaagg tggaaatcaa gcgtacggtg gccgccccct ccgtgttcat cttccccccc 420 tccgacgagc agctgaagtc cggcaccgcc tccgtggtgt gcctgctgaa taacttctac 480 cccagagagg ccaaggtgca gtggaaggtg gacaacgccc tgcagtccgg gaactcccag 540 gagagcgtga ccgagcagga cagcaaggac agcacctaca gcctgagcag caccctgacc 600 ctgagcaaag ccgactacga gaagcacaag gtgtacgcct gcgaggtgac ccaccagggc 660 ctgagctccc ccgtcaccaa gagcttcaac aggggggagt gttag 705 <210> 56 <211> 234 <212> PRT <213> artificial <220> <223> Amino Acid Sequence of hR198_LG1 <400> 56 Met Val Leu Gln Thr Gln Val Phe Ile Ser Leu Leu Leu Trp Ile Ser 1 5 10 15 Gly Ala Tyr Gly Asp Ile Gln Leu Thr Gln Ser Ser Ser Ser Leu Ser             20 25 30 Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser         35 40 45 Ile Gly Gly Ser Leu Ser Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro     50 55 60 Lys Arg Leu Ile Tyr Ser Thr Ser Thr Leu Glu Ser Gly Val Ser Ser 65 70 75 80 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser                 85 90 95 Ser Leu Gln Pro Glu Asp Phe Ala Met Tyr Tyr Cys Leu Gln Phe Ala             100 105 110 Ile Phe Pro Asp Ser Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg         115 120 125 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln     130 135 140 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 145 150 155 160 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser                 165 170 175 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr             180 185 190 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys         195 200 205 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro     210 215 220 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 <210> 57 <211> 705 <212> DNA <213> artificial <220> <223> Nucleotide Sequence coding hR198_LG2 <400> 57 atggtgctgc agacccaggt gttcatctcc ctgctgctgt ggatctccgg cgcgtacggc 60 gatatccagc tgacccagag ccctagcagc ctgtctgcca gcgtgggcga cagagtgacc 120 atcacctgtc acgccagcca gaacatcggc ggcagcctga gctggttcca gcagaaaccc 180 ggcaaggccc ccaagcggct gatctacctg accagcaccc tggactgggg cgtgcccagc 240 agattttctg gcagcggctc cggcaccgac tacaccctga caatcagcag cctgcagccc 300 gaggacttcg ccatgtacta ctgcctgcag ttcgccatct tccccgacag ctttggccag 360 ggcaccaagg tggaaatcaa gcgtacggtg gccgccccct ccgtgttcat cttccccccc 420 tccgacgagc agctgaagtc cggcaccgcc tccgtggtgt gcctgctgaa taacttctac 480 cccagagagg ccaaggtgca gtggaaggtg gacaacgccc tgcagtccgg gaactcccag 540 gagagcgtga ccgagcagga cagcaaggac agcacctaca gcctgagcag caccctgacc 600 ctgagcaaag ccgactacga gaagcacaag gtgtacgcct gcgaggtgac ccaccagggc 660 ctgagctccc ccgtcaccaa gagcttcaac aggggggagt gttag 705 <210> 58 <211> 234 <212> PRT <213> artificial <220> <223> Amino Acid Sequence of hR198_LG2 <400> 58 Met Val Leu Gln Thr Gln Val Phe Ile Ser Leu Leu Leu Trp Ile Ser 1 5 10 15 Gly Ala Tyr Gly Asp Ile Gln Leu Thr Gln Ser Ser Ser Ser Leu Ser             20 25 30 Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys His Ala Ser Gln Asn         35 40 45 Ile Gly Gly Ser Leu Ser Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro     50 55 60 Lys Arg Leu Ile Tyr Leu Thr Ser Thr Leu Asp Trp Gly Val Ser Ser 65 70 75 80 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser                 85 90 95 Ser Leu Gln Pro Glu Asp Phe Ala Met Tyr Tyr Cys Leu Gln Phe Ala             100 105 110 Ile Phe Pro Asp Ser Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg         115 120 125 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln     130 135 140 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 145 150 155 160 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser                 165 170 175 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr             180 185 190 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys         195 200 205 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro     210 215 220 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 <210> 59 <211> 705 <212> DNA <213> artificial <220> <223> Nucleotide Sequence coding hR198_LG3 <400> 59 atggtgctgc agacccaggt gttcatctcc ctgctgctgt ggatctccgg cgcgtacggc 60 gatatccagc tgacccagag ccctagcagc ctgtctgcca gcgtgggcga cagagtgacc 120 atcacctgtc acgccagccg gaacatcggc ggcagcctga gctggttcca gcagaaaccc 180 ggcaaggccc ccaagcggct gatctacctg accagcagcc tggactgggg cgtgcccagc 240 agattttctg gcagcggctc cggcaccgac tacaccctga caatcagcag cctgcagccc 300 gaggacttcg ccatgtacta ctgcctgcag ttcgccatct tccccgacag ctttggccag 360 ggcaccaagg tggaaatcaa gcgtacggtg gccgccccct ccgtgttcat cttccccccc 420 tccgacgagc agctgaagtc cggcaccgcc tccgtggtgt gcctgctgaa taacttctac 480 cccagagagg ccaaggtgca gtggaaggtg gacaacgccc tgcagtccgg gaactcccag 540 gagagcgtga ccgagcagga cagcaaggac agcacctaca gcctgagcag caccctgacc 600 ctgagcaaag ccgactacga gaagcacaag gtgtacgcct gcgaggtgac ccaccagggc 660 ctgagctccc ccgtcaccaa gagcttcaac aggggggagt gttag 705 <210> 60 <211> 234 <212> PRT <213> artificial <220> <223> Amino Acid Sequence of hR198_LG3 <400> 60 Met Val Leu Gln Thr Gln Val Phe Ile Ser Leu Leu Leu Trp Ile Ser 1 5 10 15 Gly Ala Tyr Gly Asp Ile Gln Leu Thr Gln Ser Ser Ser Ser Leu Ser             20 25 30 Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys His Ala Ser Arg Asn         35 40 45 Ile Gly Gly Ser Leu Ser Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro     50 55 60 Lys Arg Leu Ile Tyr Leu Thr Ser Ser Leu Asp Trp Gly Val Ser Ser 65 70 75 80 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser                 85 90 95 Ser Leu Gln Pro Glu Asp Phe Ala Met Tyr Tyr Cys Leu Gln Phe Ala             100 105 110 Ile Phe Pro Asp Ser Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg         115 120 125 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln     130 135 140 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 145 150 155 160 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser                 165 170 175 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr             180 185 190 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys         195 200 205 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro     210 215 220 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 <210> 61 <211> 1401 <212> DNA <213> artificial <220> <223> Nucleotide Sequence coding hR198_HG1 <400> 61 atgaaacacc tgtggttctt cctcctgctg gtggcagctc ccagatgggt gctgagccag 60 gtgcagctgg tgcagtctgg cgccgaagtg aagaaaccag gcgccagcgt gaaggtgtcc 120 tgcaaggcct ctggctaccc cgtgaccgcc tactacatca gctgggtcag acaggcccca 180 ggccagggcc tggaatacgt gggctacatc gacatgggca acggccggac cgactacaac 240 gcccggttta agggcagagc caccctgacc gtggacaaga gcaccagcac cgcctacatg 300 gaactgagca gcctgcggag cgaggacacc gccgtgtact actgcgccag agacagcaac 360 tggggcgccg actattgggg ccagggcaca ctcgtgaccg tcagctcagc ctccaccaag 420 ggcccaagcg tcttccccct ggcaccctcc tccaagagca cctctggcgg cacagccgcc 480 ctgggctgcc tggtcaagga ctacttcccc gaacccgtga ccgtgagctg gaactcaggc 540 gccctgacca gcggcgtgca caccttcccc gctgtcctgc agtcctcagg actctactcc 600 ctcagcagcg tggtgaccgt gccctccagc agcttgggca cccagaccta catctgcaac 660 gtgaatcaca agcccagcaa caccaaggtg gacaagagag ttgagcccaa atcttgtgac 720 aaaactcaca catgcccacc ctgcccagca cctgaactcc tggggggacc ctcagtcttc 780 ctcttccccc caaaacccaa ggacaccctc atgatctccc ggacccctga ggtcacatgc 840 gtggtggtgg acgtgagcca cgaagaccct gaggtcaagt tcaactggta cgtggacggc 900 gtggaggtgc ataatgccaa gacaaagccc cgggaggagc agtacaacag cacgtaccgg 960 gtggtcagcg tcctcaccgt cctgcaccag gactggctta atggcaagga gtacaagtgc 1020 aaggtctcca acaaagccct cccagccccc atcgagaaaa ccatctccaa agccaaaggc 1080 cagccccggg aaccacaggt gtacaccctg cccccatccc gggaggagat gaccaagaac 1140 caggtcagcc tgacctgcct ggtcaaaggc ttctatccca gcgacatcgc cgtggagtgg 1200 gagagcaatg gccagcccga gaacaactac aagaccaccc ctcccgtgct ggactccgac 1260 ggctccttct tcctctacag caagctcacc gtggacaaga gcaggtggca gcagggcaac 1320 gtcttctcat gctccgtgat gcatgaggct ctgcacaacc actacaccca gaagagcctc 1380 tccctgtctc ccggcaaatg a 1401 <210> 62 <211> 466 <212> PRT <213> artificial <220> <223> Amino Acid Sequence of hR198_HG1 <400> 62 Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala Pro Arg Trp 1 5 10 15 Val Leu Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys             20 25 30 Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Pro Val         35 40 45 Thr Ala Tyr Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu     50 55 60 Glu Tyr Val Gly Tyr Ile Asp Met Gly Asn Gly Arg Thr Asp Tyr Asn 65 70 75 80 Ala Arg Phe Lys Gly Arg Ala Thr Leu Thr Val Asp Lys Ser Thr Ser                 85 90 95 Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val             100 105 110 Tyr Tyr Cys Ala Arg Asp Ser Asn Trp Gly Ala Asp Tyr Trp Gly Gln         115 120 125 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Ser Ser Val     130 135 140 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 145 150 155 160 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser                 165 170 175 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val             180 185 190 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro         195 200 205 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys     210 215 220 Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp 225 230 235 240 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly                 245 250 255 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile             260 265 270 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu         275 280 285 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His     290 295 300 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 305 310 315 320 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys                 325 330 335 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu             340 345 350 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr         355 360 365 Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu     370 375 380 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 385 390 395 400 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val                 405 410 415 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp             420 425 430 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His         435 440 445 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro     450 455 460 Gly Lys 465 <210> 63 <211> 1401 <212> DNA <213> artificial <220> <223> Nucleotide Sequence coding hR198_HG2 <400> 63 atgaaacacc tgtggttctt cctcctgctg gtggcagctc ccagatgggt gctgagccag 60 gtgcagctgg tgcagtctgg cgccgaagtg aagaaaccag gcgccagcgt gaaggtgtcc 120 tgcaaggcct ctggctaccc catcaccgcc tactacatca gctgggtcag acaggcccca 180 ggccagggcc tggaatacgt gggctacatc gacatgggca acggccggac cgactacaac 240 ggccggttta agggcagagc caccctgacc gtggacaaga gcaccagcac cgcctacatg 300 gaactgagca gcctgcggag cgaggacacc gccgtgtact actgcgccag agacagcaac 360 tggggcgccg actattgggg ccagggcaca ctcgtgaccg tcagctcagc ctccaccaag 420 ggcccaagcg tcttccccct ggcaccctcc tccaagagca cctctggcgg cacagccgcc 480 ctgggctgcc tggtcaagga ctacttcccc gaacccgtga ccgtgagctg gaactcaggc 540 gccctgacca gcggcgtgca caccttcccc gctgtcctgc agtcctcagg actctactcc 600 ctcagcagcg tggtgaccgt gccctccagc agcttgggca cccagaccta catctgcaac 660 gtgaatcaca agcccagcaa caccaaggtg gacaagagag ttgagcccaa atcttgtgac 720 aaaactcaca catgcccacc ctgcccagca cctgaactcc tggggggacc ctcagtcttc 780 ctcttccccc caaaacccaa ggacaccctc atgatctccc ggacccctga ggtcacatgc 840 gtggtggtgg acgtgagcca cgaagaccct gaggtcaagt tcaactggta cgtggacggc 900 gtggaggtgc ataatgccaa gacaaagccc cgggaggagc agtacaacag cacgtaccgg 960 gtggtcagcg tcctcaccgt cctgcaccag gactggctta atggcaagga gtacaagtgc 1020 aaggtctcca acaaagccct cccagccccc atcgagaaaa ccatctccaa agccaaaggc 1080 cagccccggg aaccacaggt gtacaccctg cccccatccc gggaggagat gaccaagaac 1140 caggtcagcc tgacctgcct ggtcaaaggc ttctatccca gcgacatcgc cgtggagtgg 1200 gagagcaatg gccagcccga gaacaactac aagaccaccc ctcccgtgct ggactccgac 1260 ggctccttct tcctctacag caagctcacc gtggacaaga gcaggtggca gcagggcaac 1320 gtcttctcat gctccgtgat gcatgaggct ctgcacaacc actacaccca gaagagcctc 1380 tccctgtctc ccggcaaatg a 1401 <210> 64 <211> 466 <212> PRT <213> artificial <220> <223> Amino Acid Sequence of hR198_HG2 <400> 64 Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala Pro Arg Trp 1 5 10 15 Val Leu Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys             20 25 30 Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Pro Ile         35 40 45 Thr Ala Tyr Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu     50 55 60 Glu Tyr Val Gly Tyr Ile Asp Met Gly Asn Gly Arg Thr Asp Tyr Asn 65 70 75 80 Gly Arg Phe Lys Gly Arg Ala Thr Leu Thr Val Asp Lys Ser Thr Ser                 85 90 95 Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val             100 105 110 Tyr Tyr Cys Ala Arg Asp Ser Asn Trp Gly Ala Asp Tyr Trp Gly Gln         115 120 125 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Ser Ser Val     130 135 140 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 145 150 155 160 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser                 165 170 175 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val             180 185 190 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro         195 200 205 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys     210 215 220 Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp 225 230 235 240 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly                 245 250 255 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile             260 265 270 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu         275 280 285 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His     290 295 300 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 305 310 315 320 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys                 325 330 335 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu             340 345 350 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr         355 360 365 Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu     370 375 380 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 385 390 395 400 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val                 405 410 415 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp             420 425 430 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His         435 440 445 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro     450 455 460 Gly Lys 465 <210> 65 <211> 1401 <212> DNA <213> artificial <220> <223> Nucleotide Sequence coding hR198_HG3 <400> 65 atgaaacacc tgtggttctt cctcctgctg gtggcagctc ccagatgggt gctgagccag 60 gtgcagctgg tgcagtctgg cgccgaagtg aagaaaccag gcgccagcgt gaaggtgtcc 120 tgcaaggcct ctggctaccc catcaccgcc tactacatca gctgggtcag acaggcccca 180 ggccagggcc tggaatacgt gggctacatc gacatgggca acggccggac cgactacaac 240 atgcggttta agggcagagc caccctgacc gtggacaaga gcaccagcac cgcctacatg 300 gaactgagca gcctgcggag cgaggacacc gccgtgtact actgcgccag agacagcaac 360 tggggcgccg actattgggg ccagggcaca ctcgtgaccg tcagctcagc ctccaccaag 420 ggcccaagcg tcttccccct ggcaccctcc tccaagagca cctctggcgg cacagccgcc 480 ctgggctgcc tggtcaagga ctacttcccc gaacccgtga ccgtgagctg gaactcaggc 540 gccctgacca gcggcgtgca caccttcccc gctgtcctgc agtcctcagg actctactcc 600 ctcagcagcg tggtgaccgt gccctccagc agcttgggca cccagaccta catctgcaac 660 gtgaatcaca agcccagcaa caccaaggtg gacaagagag ttgagcccaa atcttgtgac 720 aaaactcaca catgcccacc ctgcccagca cctgaactcc tggggggacc ctcagtcttc 780 ctcttccccc caaaacccaa ggacaccctc atgatctccc ggacccctga ggtcacatgc 840 gtggtggtgg acgtgagcca cgaagaccct gaggtcaagt tcaactggta cgtggacggc 900 gtggaggtgc ataatgccaa gacaaagccc cgggaggagc agtacaacag cacgtaccgg 960 gtggtcagcg tcctcaccgt cctgcaccag gactggctta atggcaagga gtacaagtgc 1020 aaggtctcca acaaagccct cccagccccc atcgagaaaa ccatctccaa agccaaaggc 1080 cagccccggg aaccacaggt gtacaccctg cccccatccc gggaggagat gaccaagaac 1140 caggtcagcc tgacctgcct ggtcaaaggc ttctatccca gcgacatcgc cgtggagtgg 1200 gagagcaatg gccagcccga gaacaactac aagaccaccc ctcccgtgct ggactccgac 1260 ggctccttct tcctctacag caagctcacc gtggacaaga gcaggtggca gcagggcaac 1320 gtcttctcat gctccgtgat gcatgaggct ctgcacaacc actacaccca gaagagcctc 1380 tccctgtctc ccggcaaatg a 1401 <210> 66 <211> 466 <212> PRT <213> artificial <220> <223> Amino Acid Sequence of hR198_HG3 <400> 66 Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala Pro Arg Trp 1 5 10 15 Val Leu Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys             20 25 30 Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Pro Ile         35 40 45 Thr Ala Tyr Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu     50 55 60 Glu Tyr Val Gly Tyr Ile Asp Met Gly Asn Gly Arg Thr Asp Tyr Asn 65 70 75 80 Met Arg Phe Lys Gly Arg Ala Thr Leu Thr Val Asp Lys Ser Thr Ser                 85 90 95 Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val             100 105 110 Tyr Tyr Cys Ala Arg Asp Ser Asn Trp Gly Ala Asp Tyr Trp Gly Gln         115 120 125 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Ser Ser Val     130 135 140 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 145 150 155 160 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser                 165 170 175 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val             180 185 190 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro         195 200 205 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys     210 215 220 Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp 225 230 235 240 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly                 245 250 255 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile             260 265 270 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu         275 280 285 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His     290 295 300 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 305 310 315 320 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys                 325 330 335 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu             340 345 350 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr         355 360 365 Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu     370 375 380 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 385 390 395 400 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val                 405 410 415 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp             420 425 430 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His         435 440 445 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro     450 455 460 Gly Lys 465 <210> 67 <211> 1401 <212> DNA <213> artificial <220> <223> Nucleotide Sequence coding hR198_H4-LALA <400> 67 atgaaacacc tgtggttctt cctcctgctg gtggcagctc ccagatgggt gctgagccag 60 gtgcagctgg tgcagtctgg cgccgaagtg aagaaaccag gcgccagcgt gaaggtgtcc 120 tgcaaggcct ctggctaccc cgtgaccgcc tactacatca gctggatcag acaggcccca 180 ggccagggcc tggaatacgt gggctacatc gacatgggca acggccggac caactacaac 240 gcccggttta agggcagagc caccctgacc gtggacaaga gcaccagcac cgcctacatg 300 gaactgagca gcctgcggag cgaggacacc gccgtgtact actgcgccag agacagcaac 360 tggggcgtgg actattgggg ccagggcaca ctcgtgaccg tcagctcagc ctccaccaag 420 ggcccaagcg tcttccccct ggcaccctcc tccaagagca cctctggcgg cacagccgcc 480 ctgggctgcc tggtcaagga ctacttcccc gaacccgtga ccgtgagctg gaactcaggc 540 gccctgacca gcggcgtgca caccttcccc gctgtcctgc agtcctcagg actctactcc 600 ctcagcagcg tggtgaccgt gccctccagc agcttgggca cccagaccta catctgcaac 660 gtgaatcaca agcccagcaa caccaaggtg gacaagagag ttgagcccaa atcttgtgac 720 aaaactcaca catgcccacc ctgcccagca cctgaagccg cggggggacc ctcagtcttc 780 ctcttccccc caaaacccaa ggacaccctc atgatctccc ggacccctga ggtcacatgc 840 gtggtggtgg acgtgagcca cgaagaccct gaggtcaagt tcaactggta cgtggacggc 900 gtggaggtgc ataatgccaa gacaaagccc cgggaggagc agtacaacag cacgtaccgg 960 gtggtcagcg tcctcaccgt cctgcaccag gactggctta atggcaagga gtacaagtgc 1020 aaggtctcca acaaagccct cccagccccc atcgagaaaa ccatctccaa agccaaaggc 1080 cagccccggg aaccacaggt gtacaccctg cccccatccc gggaggagat gaccaagaac 1140 caggtcagcc tgacctgcct ggtcaaaggc ttctatccca gcgacatcgc cgtggagtgg 1200 gagagcaatg gccagcccga gaacaactac aagaccaccc ctcccgtgct ggactccgac 1260 ggctccttct tcctctacag caagctcacc gtggacaaga gcaggtggca gcagggcaac 1320 gtcttctcat gctccgtgat gcatgaggct ctgcacaacc actacaccca gaagagcctc 1380 tccctgtctc ccggcaaatg a 1401 <210> 68 <211> 466 <212> PRT <213> artificial <220> <223> Amino Acid Sequence of hR198_H4-LALA <400> 68 Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala Pro Arg Trp 1 5 10 15 Val Leu Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys             20 25 30 Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Pro Val         35 40 45 Thr Ala Tyr Ile Ser Trp Ile Arg Gln Ala Pro Gly Gln Gly Leu     50 55 60 Glu Tyr Val Gly Tyr Ile Asp Met Gly Asn Gly Arg Thr Asn Tyr Asn 65 70 75 80 Ala Arg Phe Lys Gly Arg Ala Thr Leu Thr Val Asp Lys Ser Thr Ser                 85 90 95 Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val             100 105 110 Tyr Tyr Cys Ala Arg Asp Ser Asn Trp Gly Val Asp Tyr Trp Gly Gln         115 120 125 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Ser Ser Val     130 135 140 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 145 150 155 160 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser                 165 170 175 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val             180 185 190 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro         195 200 205 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys     210 215 220 Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp 225 230 235 240 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly                 245 250 255 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile             260 265 270 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu         275 280 285 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His     290 295 300 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 305 310 315 320 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys                 325 330 335 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu             340 345 350 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr         355 360 365 Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu     370 375 380 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 385 390 395 400 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val                 405 410 415 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp             420 425 430 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His         435 440 445 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro     450 455 460 Gly Lys 465 <210> 69 <211> 1401 <212> DNA <213> artificial <220> <223> Nucleotide Sequence coding hR198_HG1-LALA <400> 69 atgaaacacc tgtggttctt cctcctgctg gtggcagctc ccagatgggt gctgagccag 60 gtgcagctgg tgcagtctgg cgccgaagtg aagaaaccag gcgccagcgt gaaggtgtcc 120 tgcaaggcct ctggctaccc cgtgaccgcc tactacatca gctgggtcag acaggcccca 180 ggccagggcc tggaatacgt gggctacatc gacatgggca acggccggac cgactacaac 240 gcccggttta agggcagagc caccctgacc gtggacaaga gcaccagcac cgcctacatg 300 gaactgagca gcctgcggag cgaggacacc gccgtgtact actgcgccag agacagcaac 360 tggggcgccg actattgggg ccagggcaca ctcgtgaccg tcagctcagc ctccaccaag 420 ggcccaagcg tcttccccct ggcaccctcc tccaagagca cctctggcgg cacagccgcc 480 ctgggctgcc tggtcaagga ctacttcccc gaacccgtga ccgtgagctg gaactcaggc 540 gccctgacca gcggcgtgca caccttcccc gctgtcctgc agtcctcagg actctactcc 600 ctcagcagcg tggtgaccgt gccctccagc agcttgggca cccagaccta catctgcaac 660 gtgaatcaca agcccagcaa caccaaggtg gacaagagag ttgagcccaa atcttgtgac 720 aaaactcaca catgcccacc ctgcccagca cctgaagccg cggggggacc ctcagtcttc 780 ctcttccccc caaaacccaa ggacaccctc atgatctccc ggacccctga ggtcacatgc 840 gtggtggtgg acgtgagcca cgaagaccct gaggtcaagt tcaactggta cgtggacggc 900 gtggaggtgc ataatgccaa gacaaagccc cgggaggagc agtacaacag cacgtaccgg 960 gtggtcagcg tcctcaccgt cctgcaccag gactggctta atggcaagga gtacaagtgc 1020 aaggtctcca acaaagccct cccagccccc atcgagaaaa ccatctccaa agccaaaggc 1080 cagccccggg aaccacaggt gtacaccctg cccccatccc gggaggagat gaccaagaac 1140 caggtcagcc tgacctgcct ggtcaaaggc ttctatccca gcgacatcgc cgtggagtgg 1200 gagagcaatg gccagcccga gaacaactac aagaccaccc ctcccgtgct ggactccgac 1260 ggctccttct tcctctacag caagctcacc gtggacaaga gcaggtggca gcagggcaac 1320 gtcttctcat gctccgtgat gcatgaggct ctgcacaacc actacaccca gaagagcctc 1380 tccctgtctc ccggcaaatg a 1401 <210> 70 <211> 466 <212> PRT <213> artificial <220> <223> Amino Acid Sequence of hR198_HG1-LALA <400> 70 Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala Pro Arg Trp 1 5 10 15 Val Leu Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys             20 25 30 Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Pro Val         35 40 45 Thr Ala Tyr Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu     50 55 60 Glu Tyr Val Gly Tyr Ile Asp Met Gly Asn Gly Arg Thr Asp Tyr Asn 65 70 75 80 Ala Arg Phe Lys Gly Arg Ala Thr Leu Thr Val Asp Lys Ser Thr Ser                 85 90 95 Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val             100 105 110 Tyr Tyr Cys Ala Arg Asp Ser Asn Trp Gly Ala Asp Tyr Trp Gly Gln         115 120 125 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Ser Ser Val     130 135 140 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 145 150 155 160 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser                 165 170 175 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val             180 185 190 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro         195 200 205 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys     210 215 220 Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp 225 230 235 240 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly                 245 250 255 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile             260 265 270 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu         275 280 285 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His     290 295 300 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 305 310 315 320 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys                 325 330 335 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu             340 345 350 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr         355 360 365 Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu     370 375 380 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 385 390 395 400 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val                 405 410 415 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp             420 425 430 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His         435 440 445 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro     450 455 460 Gly Lys 465 <210> 71 <211> 1118 <212> DNA <213> artificial <220> <223> Nucleotide Sequence coding Human Heavy Chain Secretory Signal and        Human IgG2 Constant Region <400> 71 gcctccggac tctagagcca ccatgaaaca cctgtggttc ttcctcctgc tggtggcagc 60 tcccagatgg gtgctgagcc aggtgcaatt gtgcaggcgg ttagctcagc cagcaccaag 120 ggcccttccg tgttccctct ggccccttgt agccgttcca ccagcgagtc caccgccgcc 180 cttggctgtc tggtgaagga ctacttccct gagcctgtga ccgtgagctg gaactccgga 240 gcccttacca gcggcgtgca caccttccct gccgtgctgc agtccagcgg cctttactcc 300 ctgagctccg tggtgaccgt gcctagctcc aacttcggca cccaaaccta cacctgtaac 360 gtggaccaca agcctagcaa caccaaggtg gacaagaccg tggagcgtaa gtgttgtgtg 420 gagtgtcctc cttgtcctgc ccctcctgtg gccggacctt ccgtgttcct tttccctcct 480 aagcctaagg acaccctgat gatcagccgt acccctgagg tgacctgtgt ggtggtggac 540 gtgtcccacg aggaccctga ggtgcagttc aactggtacg tggacggcgt ggaggtgcac 600 aacgccaaga ccaagcctcg tgaggagcaa ttcaacagca ccttccgtgt ggtgtccgtg 660 cttaccgtgg tgcaccaaga ctggctgaac ggcaaggagt acaagtgtaa ggtgagcaac 720 aagggacttc ctgcccctat cgagaagacc atctccaaga ccaagggcca acctcgtgag 780 cctcaagtgt acacccttcc tcctagccgt gaggagatga ccaagaacca agtgtccctt 840 acctgtctgg tgaagggctt ctaccctagc gacatcgccg tggagtggga gtccaacgga 900 caacctgaga acaactacaa gaccacccct cctatgcttg acagcgacgg ctccttcttc 960 ctgtacagca agctgaccgt ggacaagtcc cgttggcaac aaggcaacgt gttcagctgt 1020 tccgtgatgc acgaggccct gcacaaccac tacacccaaa agagcctttc cctgagccct 1080 ggaaagtgat atcgggcccg tttaaacggg ggaggcta 1118 <210> 72 <211> 1395 <212> DNA <213> artificial <220> <223> Nucleotide Sequence coding Heavy Chain of 2C1.1 <400> 72 atgaaacacc tgtggttctt cctcctgctg gtggcagctc ccagatgggt gctgagccag 60 gtgcagctgc agcagtgggg agccggcctg ctgaagccca gcgagacact gagcctgacc 120 tgcgctgtgt acggaggcag cttcagcggc tactactggt cctggatccg gcagccccct 180 ggcaagggcc tggaatggat cggcgagatc gaccacagcg gcagcaccaa ctacaacccc 240 gccctgaagt cccggctgac catcagcgtg gacaccagca agaaccagtt cagcctgaag 300 ctgagcagcg tgacagccgc cgacaccgcc gtgtactact gtgccagagc cggcagcggc 360 ggctacgagg attggttcga tccttggggc cagggcaccc tggtgaccgt cagctcagcc 420 agcaccaagg gcccttccgt gttccctctg gccccttgta gccgttccac cagcgagtcc 480 accgccgccc ttggctgtct ggtgaaggac tacttccctg agcctgtgac cgtgagctgg 540 aactccggag cccttaccag cggcgtgcac accttccctg ccgtgctgca gtccagcggc 600 ctttactccc tgagctccgt ggtgaccgtg cctagctcca acttcggcac ccaaacctac 660 acctgtaacg tggaccacaa gcctagcaac accaaggtgg acaagaccgt ggagcgtaag 720 tgttgtgtgg agtgtcctcc ttgtcctgcc cctcctgtgg ccggaccttc cgtgttcctt 780 ttccctccta agcctaagga caccctgatg atcagccgta cccctgaggt gacctgtgtg 840 gtggtggacg tgtcccacga ggaccctgag gtgcagttca actggtacgt ggacggcgtg 900 gaggtgcaca acgccaagac caagcctcgt gaggagcaat tcaacagcac cttccgtgtg 960 gtgtccgtgc ttaccgtggt gcaccaagac tggctgaacg gcaaggagta caagtgtaag 1020 gtgagcaaca agggacttcc tgcccctatc gagaagacca tctccaagac caagggccaa 1080 cctcgtgagc ctcaagtgta cacccttcct cctagccgtg aggagatgac caagaaccaa 1140 gtgtccctta cctgtctggt gaagggcttc taccctagcg acatcgccgt ggagtgggag 1200 tccaacggac aacctgagaa caactacaag accacccctc ctatgcttga cagcgacggc 1260 tccttcttcc tgtacagcaa gctgaccgtg gacaagtccc gttggcaaca aggcaacgtg 1320 ttcagctgtt ccgtgatgca cgaggccctg cacaaccact acacccaaaa gagcctttcc 1380 ctgagccctg gaaag 1395 <210> 73 <211> 465 <212> PRT <213> artificial <220> <223> Amino Acid Sequence of Heavy Chain of 2C1.1 <400> 73 Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala Pro Arg Trp 1 5 10 15 Val Leu Ser Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys             20 25 30 Pro Ser Glu Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe         35 40 45 Ser Gly Tyr Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu     50 55 60 Glu Trp Ile Gly Glu Ile Asp His Ser Gly Ser Thr Asn Tyr Asn Pro 65 70 75 80 Ala Leu Lys Ser Arg Leu Thr Ile Ser Val Asp Thr Ser Lys Asn Gln                 85 90 95 Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr             100 105 110 Tyr Cys Ala Arg Ala Gly Ser Gly Gly Tyr Glu Asp Trp Phe Asp Pro         115 120 125 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly     130 135 140 Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser 145 150 155 160 Thr Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val                 165 170 175 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe             180 185 190 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val         195 200 205 Thr Val Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val     210 215 220 Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys 225 230 235 240 Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro                 245 250 255 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser             260 265 270 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Ser Glu Asp         275 280 285 Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn     290 295 300 Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val 305 310 315 320 Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu                 325 330 335 Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys             340 345 350 Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr         355 360 365 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr     370 375 380 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 385 390 395 400 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu                 405 410 415 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys             420 425 430 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu         435 440 445 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly     450 455 460 Lys 465 <210> 74 <211> 739 <212> DNA <213> artificial <220> <223> Nucleotide Sequence coding Light Chain of 2C1.1 <400> 74 atggtgctgc agacccaggt gttcatctcc ctgctgctgt ggatctccgg cgcgtacggc 60 cagtccgtgc tgacccagcc tccttccgtg tctggcgccc ctggccagag agtgaccatc 120 agctgtaccg gcagcagcag caacatcgga gccggctaca acgtgcactg gtatcagcag 180 ttcccccgga ccgaccccaa gctgctgatc tacgtgtaca acatccggcc cagcggcgtg 240 cccgaccggt tttctggcag cagaagcggc acaagcgcca gcctggccat caccggcctg 300 cagaccgagg acgaggccga ctactactgc cagagctacg acagcagcct gagcggcgtg 360 gtgttcggcg gaggcaccaa gctgacagtg ctgggccagc ccaaggccaa ccccaccgtg 420 accctgttcc ccccaagcag cgaggaactg caggccaaca aggccaccct ggtgtgcctg 480 atcagcgact tctaccctgg cgccgtgaca gtggcctgga aggccgatgg atctcccgtg 540 aaggccggcg tggaaaccac caagcccagc aagcagagca acaacaaata cgccgccagc 600 agctacctga gcctgacccc cgagcagtgg aagtcccacc ggtcctacag ctgccaggtg 660 acacacgagg gcagcaccgt ggaaaagacc gtggccccca ccgagtgcag ctaggggccc 720 gtttaaacgg gggaggcta 739 <210> 75 <211> 237 <212> PRT <213> artificial <220> <223> Amino Acid Sequence of Light Chain of 2C1.1 <400> 75 Met Val Leu Gln Thr Gln Val Phe Ile Ser Leu Leu Leu Trp Ile Ser 1 5 10 15 Gly Ala Tyr Gly Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Gly             20 25 30 Ala Pro Gly Gln Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Asn         35 40 45 Ile Gly Ala Gly Tyr Asn Val His Trp Tyr Gln Gln Phe Pro Arg Thr     50 55 60 Asp Pro Lys Leu Leu Ile Tyr Val Tyr Asn Ile Arg Pro Ser Gly Val 65 70 75 80 Pro Asp Arg Phe Ser Gly Ser Arg Ser Gly Thr Ser Ala Ser Leu Ala                 85 90 95 Ile Thr Gly Leu Gln Thr Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser             100 105 110 Tyr Asp Ser Ser Leu Ser Gly Val Val Phe Gly Gly Gly Thr Lys Leu         115 120 125 Thr Val Leu Gly Gln Pro Lys Ala Asn Pro Thr Val Thr Leu Phe Pro     130 135 140 Pro Ser Ser Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu 145 150 155 160 Ile Ser Asp Phe Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp                 165 170 175 Gly Ser Pro Val Lys Ala Gly Val Glu Thr Thr Lys Pro Ser Lys Gln             180 185 190 Ser Asn Asn Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu         195 200 205 Gln Trp Lys Ser His Arg Ser Ser Tyr Ser Cys Gln Val Thr His Glu Gly     210 215 220 Ser Thr Val Glu Lys Thr Val Ala Pro Thr Glu Cys Ser 225 230 235 <210> 76 <211> 1395 <212> DNA <213> artificial <220> <223> Nucleotide Sequence coding Heavy Chain of 5H3.1 <400> 76 atgaaacacc tgtggttctt cctcctgctg gtggcagctc ccagatgggt gctgagccag 60 gtgcagctgg tgcagtctgg cgccgaagtg aagaaacctg gcgctagcgt gaaggtgtcc 120 tgcaaggcca gcggctacac cttcaccgac tactacatga actgggtgcg ccaggctcca 180 ggacagggcc tggaatggat gggctggatc aaccccaaca gcggcggcac caaatacgcc 240 cagaaattcc agggcagagt gaccatgacc cgggacacca gcatccggac cgcctacatg 300 gaactgagcc ggctgagaag cgacgacacc gccgtgtact actgcgccag agagtacggc 360 ggcaacagcg attggttcga cccctggggc cagggcaccc tggtgaccgt cagctcagcc 420 agcaccaagg gcccttccgt gttccctctg gccccttgta gccgttccac cagcgagtcc 480 accgccgccc ttggctgtct ggtgaaggac tacttccctg agcctgtgac cgtgagctgg 540 aactccggag cccttaccag cggcgtgcac accttccctg ccgtgctgca gtccagcggc 600 ctttactccc tgagctccgt ggtgaccgtg cctagctcca acttcggcac ccaaacctac 660 acctgtaacg tggaccacaa gcctagcaac accaaggtgg acaagaccgt ggagcgtaag 720 tgttgtgtgg agtgtcctcc ttgtcctgcc cctcctgtgg ccggaccttc cgtgttcctt 780 ttccctccta agcctaagga caccctgatg atcagccgta cccctgaggt gacctgtgtg 840 gtggtggacg tgtcccacga ggaccctgag gtgcagttca actggtacgt ggacggcgtg 900 gaggtgcaca acgccaagac caagcctcgt gaggagcaat tcaacagcac cttccgtgtg 960 gtgtccgtgc ttaccgtggt gcaccaagac tggctgaacg gcaaggagta caagtgtaag 1020 gtgagcaaca agggacttcc tgcccctatc gagaagacca tctccaagac caagggccaa 1080 cctcgtgagc ctcaagtgta cacccttcct cctagccgtg aggagatgac caagaaccaa 1140 gtgtccctta cctgtctggt gaagggcttc taccctagcg acatcgccgt ggagtgggag 1200 tccaacggac aacctgagaa caactacaag accacccctc ctatgcttga cagcgacggc 1260 tccttcttcc tgtacagcaa gctgaccgtg gacaagtccc gttggcaaca aggcaacgtg 1320 ttcagctgtt ccgtgatgca cgaggccctg cacaaccact acacccaaaa gagcctttcc 1380 ctgagccctg gaaag 1395 <210> 77 <211> 465 <212> PRT <213> artificial <220> <223> Amino Acid Sequence of Heavy Chain of 5H3.1 <400> 77 Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala Pro Arg Trp 1 5 10 15 Val Leu Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys             20 25 30 Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe         35 40 45 Thr Asp Tyr Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu     50 55 60 Glu Trp Met Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr Lys Tyr Ala 65 70 75 80 Gln Lys Phe Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Arg                 85 90 95 Thr Ala Tyr Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val             100 105 110 Tyr Tyr Cys Ala Arg Glu Tyr Gly Gly Asn Ser Asp Trp Phe Asp Pro         115 120 125 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly     130 135 140 Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser 145 150 155 160 Thr Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val                 165 170 175 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe             180 185 190 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val         195 200 205 Thr Val Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val     210 215 220 Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys 225 230 235 240 Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro                 245 250 255 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser             260 265 270 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Ser Glu Asp         275 280 285 Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn     290 295 300 Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val 305 310 315 320 Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu                 325 330 335 Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys             340 345 350 Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr         355 360 365 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr     370 375 380 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 385 390 395 400 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu                 405 410 415 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys             420 425 430 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu         435 440 445 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly     450 455 460 Lys 465 <210> 78 <211> 742 <212> DNA <213> artificial <220> <223> Nucleotide Sequence coding Light Chain of 5H3.1 <400> 78 atggtgctgc agacccaggt gttcatctcc ctgctgctgt ggatctccgg cgcgtacggc 60 cagtccgtgc tgacccagcc tccttccgtg tctggcgccc ctggccagag agtgaccatc 120 agctgtaccg gcagcagcag caacatcgga gctggatacg acgtgcactg gtatcagcag 180 ctgcccggca ccgcccccaa gctgctgatc tacggcaaca gcaaccggcc cagcggcgtg 240 cccgatagat tcagcggcag caagagcggc accagcgcca gcctggccat taccggactg 300 caggccgagg acgaggccga ctactactgc cagagctacg acaaccggct gagcgacagc 360 gtggtgatcg gcggaggcac caagctggcc gtgcagggac agcccaaggc caaccccacc 420 gtgaccctgt tccccccaag cagcgaggaa ctgcaggcca acaaggccac cctggtgtgc 480 ctgatcagcg acttctaccc tggcgccgtg acagtggcct ggaaggccga tggatctccc 540 gtgaaggccg gcgtggaaac caccaagccc agcaagcaga gcaacaacaa atacgccgcc 600 agcagctacc tgagcctgac ccccgagcag tggaagtccc accggtccta cagctgccag 660 gtgacacacg agggcagcac cgtggaaaag accgtggccc ccaccgagtg cagctagggg 720 cccgtttaaa cgggggaggc ta 742 <210> 79 <211> 238 <212> PRT <213> artificial <220> <223> Amino Acid Sequence of Light Chain of 5H3.1 <400> 79 Met Val Leu Gln Thr Gln Val Phe Ile Ser Leu Leu Leu Trp Ile Ser 1 5 10 15 Gly Ala Tyr Gly Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Gly             20 25 30 Ala Pro Gly Gln Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Asn         35 40 45 Ile Gly Ala Gly Tyr Asp Val His His Trp Tyr Gln Gln Leu Pro Gly Thr     50 55 60 Ala Pro Lys Leu Leu Ile Tyr Gly Asn Ser Asn Arg Pro Ser Gly Val 65 70 75 80 Pro Asp Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala                 85 90 95 Ile Thr Gly Leu Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser             100 105 110 Tyr Asp Asn Arg Leu Ser Asp Ser Val Val Ile Gly Gly Gly Thr Lys         115 120 125 Leu Ala Val Gln Gly Gln Pro Lys Ala Asn Pro Thr Val Thr Leu Phe     130 135 140 Pro Pro Ser Ser Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys 145 150 155 160 Leu Ile Ser Asp Phe Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala                 165 170 175 Asp Gly Ser Pro Val Lys Ala Gly Val Glu Thr Thr Lys Pro Ser Lys             180 185 190 Gln Ser Asn Asn Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro         195 200 205 Glu Gln Trp Lys Ser His Arg Ser Tyr Ser Cys Gln Val Thr His Glu     210 215 220 Gly Ser Thr Val Glu Lys Thr Val Ala Pro Thr Glu Cys Ser 225 230 235 <210> 80 <211> 1433 <212> DNA <213> artificial <220> <223> Nucleotide Sequence coding Heavy Chain of 10F8 <400> 80 ccagcctccg gactctagag ccaccatgaa gcacctgtgg ttctttctgc tgctggtggc 60 cgctcccaga tgggtgctgt ctcaggtgca gctgcagcag tctggcgccg aactcgtgcg 120 gcctggaagc agcgtgaaga tcagctgcaa ggccagcggc tacgccttcc ggtcctactg 180 gatgaactgg gtcaagcaga ggccaggcca gggcctggaa tggatcggcc acatctatcc 240 cggcgacggc gacaccaact acaacggcaa gttcaagggc aaggccaccc tgaccgccga 300 caagagcagc agcacagcct acatgcagct gtccagcctg accagcgagg acagcgccgt 360 gtacctgtgt ggcagaggcg gcacaaccgt ggtggtggat tattggggcc agggcaccac 420 actgaccgtg tccagcgcca agaccacccc cccatctgtg tatcctctgg cccctggatc 480 tgccgcccag accaacagca tggtcaccct gggctgcctc gtgaagggct acttccctga 540 gcctgtgacc gtgacctgga acagcggctc tctgtctagc ggcgtgcaca cctttccagc 600 cgtgctgcag agcgacctgt acaccctgag cagctccgtg accgtgccta gcagcacctg 660 gcctagcgag acagtgacct gcaacgtggc ccaccctgcc agctctacca aggtggacaa 720 gaaaatcgtg ccccgggact gcggctgcaa gccctgtatc tgtaccgtgc ccgaggtgtc 780 ctccgtgttc atcttcccac ccaagcccaa ggacgtgctg accatcaccc tgacacccaa 840 agtgacatgt gtggtggtgg acatcagcaa ggacgacccc gaggtgcagt tcagttggtt 900 cgtggacgac gtggaagtgc acacagccca gacccagccc agagaggaac agttcaacag 960 caccttcaga agcgtgtccg agctgcccat catgcaccag gactggctta acggcaaaga 1020 attcaagtgc agagtgaaca gcgccgcctt ccctgccccc atcgagaaaa ccatctccaa 1080 gaccaagggc agacccaagg ccccccaggt gtacacaatc cccccaccca aagaacagat 1140 ggccaaggac aaggtgtccc tgacctgcat gatcaccgat ttcttcccag aggacatcac 1200 cgtggaatgg cagtggaacg gccagcccgc cgagaactac aagaacaccc agcctatcat 1260 ggacaccgac ggcagctact tcgtgtacag caagctgaac gtgcagaagt ccaactggga 1320 ggccggcaac accttcacct gtagcgtgct gcacgagggc ctgcacaatc accacaccga 1380 gaagtccctg tcccacagcc ccggcaaatg agtttaaacg ggggaggcta act 1433 <210> 81 <211> 461 <212> PRT <213> artificial <220> <223> Amino Acid Sequence of Heavy Chain of 10F8 <400> 81 Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala Pro Arg Trp 1 5 10 15 Val Leu Ser Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg             20 25 30 Pro Gly Ser Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe         35 40 45 Arg Ser Tyr Trp Met Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu     50 55 60 Glu Trp Ile Gly His Ile Tyr Pro Gly Asp Gly Asp Thr Asn Tyr Asn 65 70 75 80 Gly Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser                 85 90 95 Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val             100 105 110 Tyr Leu Cys Gly Arg Gly Gly Thr Thr Val Val Val Asp Tyr Trp Gly         115 120 125 Gln Gly Thr Thr Leu Thr Val Ser Ser Ala Lys Thr Thr Pro Ser Ser     130 135 140 Val Tyr Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val 145 150 155 160 Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val                 165 170 175 Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala             180 185 190 Val Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro         195 200 205 Ser Ser Thr Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala His Pro     210 215 220 Ala Ser Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly 225 230 235 240 Cys Lys Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile                 245 250 255 Phe Pro Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys             260 265 270 Val Thr Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln         275 280 285 Phe Ser Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln     290 295 300 Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu 305 310 315 320 Pro Ile Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg                 325 330 335 Val Asn Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys             340 345 350 Thr Lys Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro         355 360 365 Lys Glu Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr     370 375 380 Asp Phe Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln 385 390 395 400 Pro Ala Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly                 405 410 415 Ser Tyr Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu             420 425 430 Ala Gly Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn         435 440 445 His His Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys     450 455 460 <210> 82 <211> 770 <212> DNA <213> artificial <220> <223> Nucleotide Sequence coding Light Chain of 10F8 <400> 82 ccagcctccg gactctagag ccaccatggt gctgcagacc caggtgttca tcagcctgct 60 gctgtggatc agcggcgcct acggcgacat cgtgatgagc cagagcccta gcagcctggc 120 cgtgtctgcc ggcgagaaag tgaccatgag ctgcaagagc agccagtccc tgctgaacag 180 ccggacccgg aagaactacc tggcctggta tcagcagaag cccggccagt cccccaagct 240 gctgatctac tgggccagca ccagagaaag cggcgtgccc gatagattca ccggcagcgg 300 ctctggcacc gacttcaccc tgacaatcag cagcgtgcag gccgaggacc tggctgtgta 360 ctactgcaag cagagctaca acctgccctg gaccttcggc ggaggcacca agctggaaat 420 caagagagcc gacgccgctc ccaccgtgtc catctttcca cctagcagcg agcagctgac 480 cagcggcgga gctagcgtcg tgtgcttcct gaacaacttc taccccaagg acatcaacgt 540 gaagtggaag atcgacggca gcgagcggca gaacggcgtg ctgaatagct ggaccgacca 600 ggacagcaag gactccacct acagcatgtc cagcaccctg accctgacca aggacgagta 660 cgagcggcac aacagctaca catgcgaggc cacccacaag accagcacct cccccatcgt 720 gaagtccttc aaccggaacg agtgctgagt ttaaacgggg gaggctaact 770 <210> 83 <211> 240 <212> PRT <213> artificial <220> <223> Amino Acid Sequence of Light Chain of 10F8 <400> 83 Met Val Leu Gln Thr Gln Val Phe Ile Ser Leu Leu Leu Trp Ile Ser 1 5 10 15 Gly Ala Tyr Gly Asp Ile Val Met Ser Ser Gln Ser Pro Ser Ser Leu Ala             20 25 30 Val Ser Ala Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser         35 40 45 Leu Leu Asn Ser Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln     50 55 60 Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg 65 70 75 80 Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp                 85 90 95 Phe Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr             100 105 110 Tyr Cys Lys Gln Ser Tyr Asn Leu Pro Trp Thr Phe Gly Gly Gly Thr         115 120 125 Lys Leu Glu Ile Lys Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe     130 135 140 Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys 145 150 155 160 Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile                 165 170 175 Asp Gly Ser Glu Arg Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln             180 185 190 Asp Ser Lys Asp Ser Thr Ser Ser Met Ser Ser Thr Leu Thr Leu Thr         195 200 205 Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His     210 215 220 Lys Thr Ser Thr Ser Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys 225 230 235 240 <210> 84 <211> 1436 <212> DNA <213> artificial <220> <223> Nucleotide Sequence coding Heavy Chain of 14F74 <400> 84 ccagcctccg gactctagag ccaccatgaa gcacctgtgg ttctttctgc tgctggtggc 60 cgctcccaga tgggtgctgt ctcaggtgca gctgcagcag tctggcgccg aactcgtgcg 120 gcctggaagc agcgtgaaga tcagctgcaa ggccagcggc tacgccttca gcagctactg 180 gatgaactgg gtcaagcagc ggccaggcca gggcctggaa tggatcggcc atatctatcc 240 cggcgacggc gacaccaact acaacggcaa gttcaagggc aaggccaccc tgaccgccga 300 caagagcagc agcacagcct acatgcagct gagcggcctg accagcgagg acagcgccgt 360 gtacttctgc gccagaagcg gcagactgag attcgccatg gactactggg gccagggcac 420 cagcgtgaca gtgtctagcg ccaagaccac cccccccagc gtgtaccctc tggctcctgg 480 atctgccgcc cagaccaaca gcatggtcac cctgggctgc ctcgtgaagg gctacttccc 540 tgagcctgtg accgtgacct ggaacagcgg ctctctgtct agcggcgtgc acacctttcc 600 agccgtgctg cagagcgacc tgtacaccct gagcagctcc gtgaccgtgc ctagcagcac 660 ctggcctagc gagacagtga cctgcaacgt ggcccaccct gccagctcta ccaaggtgga 720 caagaaaatc gtgccccggg actgcggctg caagccctgt atctgtaccg tgcccgaggt 780 gtccagcgtg ttcatcttcc cacccaagcc caaggacgtg ctgaccatca ccctgacacc 840 caaagtgacc tgtgtggtgg tggacatcag caaggacgac cccgaggtgc agttcagttg 900 gttcgtggac gacgtggaag tgcacacagc ccagacccag cccagagagg aacagttcaa 960 cagcaccttc agaagcgtgt ccgagctgcc catcatgcac caggactggc tgaacggcaa 1020 agaattcaag tgcagagtga acagcgccgc cttccctgcc cccatcgaga aaaccatctc 1080 caagaccaag ggcagaccca aggcccctca ggtgtacaca atccccccac ccaaagaaca 1140 gatggccaag gacaaggtgt ccctgacctg catgatcacc gatttcttcc cagaggacat 1200 cccgtggaa tggcagtgga acggccagcc cgccgagaac tacaagaaca cccagcctat 1260 catggacacc gacggcagct acttcgtgta cagcaagctg aacgtgcaga agtccaactg 1320 ggaggccggc aacaccttca cctgtagcgt gctgcacgag ggcctgcaca atcaccacac 1380 cgagaagtcc ctgtcccaca gccccggcaa atgagtttaa acgggggagg ctaact 1436 <210> 85 <211> 462 <212> PRT <213> artificial <220> <223> Amino Acid Sequence of Heavy Chain of 14F74 <400> 85 Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala Pro Arg Trp 1 5 10 15 Val Leu Ser Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg             20 25 30 Pro Gly Ser Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe         35 40 45 Ser Ser Tyr Trp Met Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu     50 55 60 Glu Trp Ile Gly His Ile Tyr Pro Gly Asp Gly Asp Thr Asn Tyr Asn 65 70 75 80 Gly Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser                 85 90 95 Thr Ala Tyr Met Gln Leu Ser Gly Leu Thr Ser Glu Asp Ser Ala Val             100 105 110 Tyr Phe Cys Ala Arg Ser Gly Arg Leu Arg Phe Ala Met Asp Tyr Trp         115 120 125 Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala Lys Thr Thr Pro Pro     130 135 140 Ser Val Tyr Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met 145 150 155 160 Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr                 165 170 175 Val Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro             180 185 190 Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val         195 200 205 Pro Ser Ser Thr Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala His     210 215 220 Pro Ala Ser Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys 225 230 235 240 Gly Cys Lys Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe                 245 250 255 Ile Phe Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro             260 265 270 Lys Val Thr Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val         275 280 285 Gln Phe Ser Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr     290 295 300 Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Glu 305 310 315 320 Leu Pro Ile Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys                 325 330 335 Arg Val Asn Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser             340 345 350 Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro         355 360 365 Pro Lys Glu Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile     370 375 380 Thr Asp Phe Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly 385 390 395 400 Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp                 405 410 415 Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp             420 425 430 Glu Ala Gly Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His         435 440 445 Asn His His Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys     450 455 460 <210> 86 <211> 767 <212> DNA <213> artificial <220> <223> Nucleotide Sequence coding Light Chain of 14F74 <400> 86 ccagcctccg gactctagag ccaccatggt gctgcagacc caggtgttca tcagcctgct 60 gctgtggatc agcggcgcct acggcgacat cgtgatgagc cagagcccta gcagcctggc 120 cgtgtctgcc ggcgagaaag tgaccatgag ctgcaagagc agccagtccc tgctgaacag 180 ccggacccgg aagaactacc tggcctggta tcagcagaag cccggccagt cccccaagct 240 gctgatctac tgggccagca ccagagaaag cggcgtgccc gatagattca ccggcagcgg 300 ctctggcacc gacttcaccc tgacaatcag cagcgtgcag gccgaggacc tggctgtgta 360 ctactgcaag cagagctaca acctgcggac cttcggcgga ggcaccaagc tggaaatcca 420 gagagccgac gccgctccca ccgtgtccat ctttccacct agcagcgagc agctgaccag 480 cggcggagct agcgtcgtgt gcttcctgaa caacttctac cccaaggaca tcaacgtgaa 540 gtggaagatc gacggcagcg agcggcagaa cggcgtgctg aatagctgga ccgaccagga 600 cagcaaggac tccacctaca gcatgtccag caccctgacc ctgaccaagg acgagtacga 660 gcggcacaac agctacacat gcgaggccac ccacaagacc agcacctccc ccatcgtgaa 720 gtccttcaac cggaacgagt gctgagttta aacgggggag gctaact 767 <210> 87 <211> 239 <212> PRT <213> artificial <220> <223> Amino Acid Sequence of Light Chain of 14F74 <400> 87 Met Val Leu Gln Thr Gln Val Phe Ile Ser Leu Leu Leu Trp Ile Ser 1 5 10 15 Gly Ala Tyr Gly Asp Ile Val Met Ser Ser Gln Ser Pro Ser Ser Leu Ala             20 25 30 Val Ser Ala Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser         35 40 45 Leu Leu Asn Ser Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln     50 55 60 Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg 65 70 75 80 Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp                 85 90 95 Phe Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr             100 105 110 Tyr Cys Lys Gln Ser Tyr Asn Leu Arg Thr Phe Gly Gly Gly Thr Lys         115 120 125 Leu Glu Ile Gln Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro     130 135 140 Pro Ser Ser Glu Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe 145 150 155 160 Leu Asn Asn Phe Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp                 165 170 175 Gly Ser Glu Arg Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp             180 185 190 Ser Lys Asp Ser Thr Tyr Ser Ser Ser Thr Leu Thr Leu Thr Lys         195 200 205 Asp Glu Tyr Glu Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys     210 215 220 Thr Ser Thr Ser Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys 225 230 235 <210> 88 <211> 1436 <212> DNA <213> artificial <220> <223> Nucleotide Sequence coding Heavy Chain of 17F6 <400> 88 ccagcctccg gactctagag ccaccatgaa gcacctgtgg ttctttctgc tgctggtggc 60 cgctcccaga tgggtgctgt ctcaggtgca gctgcagcag tctggcgccg aactcgtgcg 120 gcctggaagc agcgtgaaga tcagctgcaa ggccagcggc tacgccttca gcagctactg 180 gatgaactgg gtcaagcagc ggccaggcca gggcctggaa tggatcggcc atatctatcc 240 cggcgacgcc gacaccaact acaacggcaa gttcaagggc aaggccaccc tgaccgccga 300 caagagcagc agcacagcct acatgcacct gtccagcctg accagcgagg acagcgccgt 360 gtacttctgc agccggcagc tgggcttcag atacgccatg gactattggg gccagggcac 420 cagcgtgacc gtgtctagcg ccaagaccac cccccctagc gtgtaccctc tggcccctgg 480 atctgccgcc cagaccaaca gcatggtcac cctgggctgc ctcgtgaagg gctacttccc 540 tgagcctgtg accgtgacct ggaacagcgg ctctctgtct agcggcgtgc acacctttcc 600 agccgtgctg cagagcgacc tgtacaccct gagcagctcc gtgacagtgc ccagctctac 660 ctggcccagc gagacagtga cctgcaacgt ggcccaccct gccagcagca ccaaggtgga 720 caagaaaatc gtgccccggg actgcggctg caagccctgt atctgtaccg tgcccgaggt 780 gtccagcgtg ttcatcttcc cacccaagcc caaggacgtg ctgaccatca ccctgacacc 840 caaagtgacc tgtgtggtgg tggacatcag caaggacgac cccgaggtgc agttcagttg 900 gttcgtggac gacgtggaag tgcacacagc ccagacccag cccagagagg aacagttcaa 960 cagcaccttc agaagcgtgt ccgagctgcc catcatgcac caggactggc tgaacggcaa 1020 agaattcaag tgcagagtga acagcgccgc cttccctgcc cccatcgaga aaaccatctc 1080 caagaccaag ggcagaccca aggcccccca ggtgtacaca atccccccac ccaaagaaca 1140 gatggccaag gacaaggtgt ccctgacctg catgatcacc gatttcttcc cagaggacat 1200 cccgtggaa tggcagtgga acggccagcc cgccgagaac tacaagaaca cccagcctat 1260 catggacacc gacggcagct acttcgtgta cagcaagctg aacgtgcaga agtccaactg 1320 ggaggccggc aacaccttca cctgtagcgt gctgcacgag ggcctgcaca atcaccacac 1380 cgagaagtcc ctgtcccaca gccccggcaa atgagtttaa acgggggagg ctaact 1436 <210> 89 <211> 462 <212> PRT <213> artificial <220> <223> Amino Acid Sequence of Heavy Chain of 17F6 <400> 89 Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala Pro Arg Trp 1 5 10 15 Val Leu Ser Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg             20 25 30 Pro Gly Ser Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe         35 40 45 Ser Ser Tyr Trp Met Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu     50 55 60 Glu Trp Ile Gly His Ile Tyr Pro Gly Asp Ala Asp Thr Asn Tyr Asn 65 70 75 80 Gly Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser                 85 90 95 Thr Ala Tyr Met His Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val             100 105 110 Tyr Phe Cys Ser Arg Gln Leu Gly Phe Arg Tyr Ala Met Asp Tyr Trp         115 120 125 Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala Lys Thr Thr Pro Pro     130 135 140 Ser Val Tyr Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met 145 150 155 160 Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr                 165 170 175 Val Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro             180 185 190 Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val         195 200 205 Pro Ser Ser Thr Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala His     210 215 220 Pro Ala Ser Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys 225 230 235 240 Gly Cys Lys Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe                 245 250 255 Ile Phe Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro             260 265 270 Lys Val Thr Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val         275 280 285 Gln Phe Ser Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr     290 295 300 Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Glu 305 310 315 320 Leu Pro Ile Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys                 325 330 335 Arg Val Asn Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser             340 345 350 Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro         355 360 365 Pro Lys Glu Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile     370 375 380 Thr Asp Phe Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly 385 390 395 400 Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp                 405 410 415 Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp             420 425 430 Glu Ala Gly Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His         435 440 445 Asn His His Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys     450 455 460 <210> 90 <211> 767 <212> DNA <213> artificial <220> <223> Nucleotide Sequence coding Light Chain of 17F6 <400> 90 ccagcctccg gactctagag ccaccatggt gctgcagacc caggtgttca tcagcctgct 60 gctgtggatc agcggcgcct acggcgacat cgtgatgagc cagagcccta gcagcctggc 120 cgtgtctgcc ggcgagaaag tgaccatgag ctgcaagagc agccagtccc tgctgaacag 180 ccggacccgg aagaactacc tggcctggta tcagcagaag cccggccagt cccccaagct 240 gctgatctac tgggccagca ccagagaaag cggcgtgccc gatagattca ccggcagcgg 300 ctctggcacc gacttcaccc tgacaatcag cagcgtgcag gccgaggacc tggctgtgta 360 ctactgcaag cagagctaca acctgcggac cttcggcgga ggcaccaagc tggaaatcaa 420 gagagccgac gccgctccca ccgtgtccat ctttccacct agcagcgagc agctgaccag 480 cggcggagct agcgtcgtgt gcttcctgaa caacttctac cccaaggaca tcaacgtgaa 540 gtggaagatc gacggcagcg agcggcagaa cggcgtgctg aatagctgga ccgaccagga 600 cagcaaggac tccacctaca gcatgtccag caccctgacc ctgaccaagg acgagtacga 660 gcggcacaac agctacacat gcgaggccac ccacaagacc agcacctccc ccatcgtgaa 720 gtccttcaac cggaacgagt gctgagttta aacgggggag gctaact 767 <210> 91 <211> 239 <212> PRT <213> artificial <220> <223> Amino Acid Sequence of Light Chain of 17F6 <400> 91 Met Val Leu Gln Thr Gln Val Phe Ile Ser Leu Leu Leu Trp Ile Ser 1 5 10 15 Gly Ala Tyr Gly Asp Ile Val Met Ser Ser Gln Ser Pro Ser Ser Leu Ala             20 25 30 Val Ser Ala Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser         35 40 45 Leu Leu Asn Ser Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln     50 55 60 Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg 65 70 75 80 Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp                 85 90 95 Phe Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr             100 105 110 Tyr Cys Lys Gln Ser Tyr Asn Leu Arg Thr Phe Gly Gly Gly Thr Lys         115 120 125 Leu Glu Ile Lys Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro     130 135 140 Pro Ser Ser Glu Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe 145 150 155 160 Leu Asn Asn Phe Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp                 165 170 175 Gly Ser Glu Arg Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp             180 185 190 Ser Lys Asp Ser Thr Tyr Ser Ser Ser Thr Leu Thr Leu Thr Lys         195 200 205 Asp Glu Tyr Glu Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys     210 215 220 Thr Ser Thr Ser Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys 225 230 235 <210> 92 <211> 11 <212> PRT <213> Rattus rattus <400> 92 Arg Ala Ser Gln Ser Ile Gly Asn Ser Leu Ser 1 5 10 <210> 93 <211> 11 <212> PRT <213> artificial <220> <223> CDRL1 of hR198_LG1 <400> 93 Arg Ala Ser Gln Ser Ile Gly Gly Ser Leu Ser 1 5 10 <210> 94 <211> 11 <212> PRT <213> artificial <220> <223> CDRL1 of hR198_LG2 <400> 94 His Ala Ser Gln Asn Ile Gly Gly Ser Leu Ser 1 5 10 <210> 95 <211> 11 <212> PRT <213> artificial <220> <223> CDRL1 of hR198_LG3 <400> 95 His Ala Ser Arg Asn Ile Gly Gly Ser Leu Ser 1 5 10 <210> 96 <211> 7 <212> PRT <213> Rattus rattus <400> 96 Ser Thr Ser Thr Leu Glu Ser 1 5 <210> 97 <211> 7 <212> PRT <213> artificial <220> <223> CDRL2 of hR198_LG2 <400> 97 Leu Thr Ser Thr Leu Asp Trp 1 5 <210> 98 <211> 7 <212> PRT <213> artificial <220> <223> CDRL2 of hR198_LG3 <400> 98 Leu Thr Ser Ser Leu Asp Trp 1 5 <210> 99 <211> 9 <212> PRT <213> Rattus rattus <400> 99 Leu Gln Phe Ala Thr Phe Pro Asp Thr 1 5 <210> 100 <211> 9 <212> PRT <213> Rattus rattus <400> 100 Leu Gln Phe Ala Thr Tyr Pro Asp Thr 1 5 <210> 101 <211> 9 <212> PRT <213> artificial <220> <223> CDRL3 of hR198_LG1 to LG3 <400> 101 Leu Gln Phe Ala Ile Phe Pro Asp Ser 1 5 <210> 102 <211> 5 <212> PRT <213> Rattus rattus <400> 102 Ala Tyr Tyr Ile Ser 1 5 <210> 103 <211> 5 <212> PRT <213> Rattus rattus <400> 103 Ser Tyr Tyr Ile Ser 1 5 <210> 104 <211> 17 <212> PRT <213> Rattus rattus <400> 104 Tyr Ile Asp Met Gly Asn Gly Arg Thr Asn Tyr Asn Ala Arg Phe Lys 1 5 10 15 Gly      <210> 105 <211> 17 <212> PRT <213> Rattus rattus <400> 105 Tyr Val Asp Met Gly Asn Gly Arg Thr Asn Tyr Asn Glu Lys Phe Lys 1 5 10 15 Gly      <210> 106 <211> 17 <212> PRT <213> artificial <220> <223> CDRH2 of hR198_HG1 <400> 106 Tyr Ile Asp Met Gly Asn Gly Arg Thr Asp Tyr Asn Ala Arg Phe Lys 1 5 10 15 Gly      <210> 107 <211> 17 <212> PRT <213> artificial <220> <223> CDRH2 of hR198_HG2 <400> 107 Tyr Ile Asp Met Gly Asn Gly Arg Thr Asp Tyr Asn Gly Arg Phe Lys 1 5 10 15 Gly      <210> 108 <211> 17 <212> PRT <213> artificial <220> <223> CDRH2 of hR198_HG3 <400> 108 Tyr Ile Asp Met Gly Asn Gly Arg Thr Asp Tyr Asn Met Arg Phe Lys 1 5 10 15 Gly      <210> 109 <211> 8 <212> PRT <213> Rattus rattus <400> 109 Asp Ser Asn Trp Gly Val Asp Tyr 1 5 <210> 110 <211> 8 <212> PRT <213> artificial <220> <223> CDRH3 of hR198_HG1 to HG3 <400> 110 Asp Ser Asn Trp Gly Ala Asp Tyr 1 5 <210> 111 <211> 1401 <212> DNA <213> artificial <220> <223> Nucleotide Sequence coding hR198_H0 <400> 111 atgaaacacc tgtggttctt cctcctgctg gtggcagctc ccagatgggt gctgagccag 60 gtgcagctgg tgcagtctgg cgccgaagtg aagaaaccag gcgccagcgt gaaggtgtcc 120 tgcaaggcct ctggctaccc cgtgaccagc tactacatca gctggatcag acaggcccca 180 ggccagggcc tggaatggat cggctatgtg gacatgggca acggccggac caactacaac 240 gagaagttca agggcagagc caccctgacc gtggacaaga gcaccagcac cgcctacatg 300 gaactgagca gcctgcggag cgaggacacc gccgtgtact actgcgccag agacagcaac 360 tggggcgtgg actattgggg ccagggcaca ctcgtgaccg tcagctcagc ctccaccaag 420 ggcccaagcg tcttccccct ggcaccctcc tccaagagca cctctggcgg cacagccgcc 480 ctgggctgcc tggtcaagga ctacttcccc gaacccgtga ccgtgagctg gaactcaggc 540 gccctgacca gcggcgtgca caccttcccc gctgtcctgc agtcctcagg actctactcc 600 ctcagcagcg tggtgaccgt gccctccagc agcttgggca cccagaccta catctgcaac 660 gtgaatcaca agcccagcaa caccaaggtg gacaagagag ttgagcccaa atcttgtgac 720 aaaactcaca catgcccacc ctgcccagca cctgaactcc tggggggacc ctcagtcttc 780 ctcttccccc caaaacccaa ggacaccctc atgatctccc ggacccctga ggtcacatgc 840 gtggtggtgg acgtgagcca cgaagaccct gaggtcaagt tcaactggta cgtggacggc 900 gtggaggtgc ataatgccaa gacaaagccc cgggaggagc agtacaacag cacgtaccgg 960 gtggtcagcg tcctcaccgt cctgcaccag gactggctta atggcaagga gtacaagtgc 1020 aaggtctcca acaaagccct cccagccccc atcgagaaaa ccatctccaa agccaaaggc 1080 cagccccggg aaccacaggt gtacaccctg cccccatccc gggaggagat gaccaagaac 1140 caggtcagcc tgacctgcct ggtcaaaggc ttctatccca gcgacatcgc cgtggagtgg 1200 gagagcaatg gccagcccga gaacaactac aagaccaccc ctcccgtgct ggactccgac 1260 ggctccttct tcctctacag caagctcacc gtggacaaga gcaggtggca gcagggcaac 1320 gtcttctcat gctccgtgat gcatgaggct ctgcacaacc actacaccca gaagagcctc 1380 tccctgtctc ccggcaaatg a 1401 <210> 112 <211> 466 <212> PRT <213> artificial <220> <223> Amino Acid Sequence of hR198_H0 <400> 112 Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala Pro Arg Trp 1 5 10 15 Val Leu Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys             20 25 30 Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Pro Val         35 40 45 Thr Ser Tyr Tyr Ile Ser Trp Ile Arg Gln Ala Pro Gly Gln Gly Leu     50 55 60 Glu Trp Ile Gly Tyr Val Asp Met Gly Asn Gly Arg Thr Asn Tyr Asn 65 70 75 80 Glu Lys Phe Lys Gly Arg Ala Thr Leu Thr Val Asp Lys Ser Thr Ser                 85 90 95 Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val             100 105 110 Tyr Tyr Cys Ala Arg Asp Ser Asn Trp Gly Val Asp Tyr Trp Gly Gln         115 120 125 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Ser Ser Val     130 135 140 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 145 150 155 160 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser                 165 170 175 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val             180 185 190 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro         195 200 205 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys     210 215 220 Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp 225 230 235 240 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly                 245 250 255 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile             260 265 270 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu         275 280 285 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His     290 295 300 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 305 310 315 320 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys                 325 330 335 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu             340 345 350 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr         355 360 365 Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu     370 375 380 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 385 390 395 400 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val                 405 410 415 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp             420 425 430 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His         435 440 445 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro     450 455 460 Gly Lys 465 <210> 113 <211> 1401 <212> DNA <213> artificial <220> <223> Nucleotide Sequence coding hR198_H5 <400> 113 atgaaacacc tgtggttctt cctcctgctg gtggcagctc ccagatgggt gctgagccag 60 gtgcagctgg tgcagtctgg cgccgaagtg aagaaaccag gcgccagcgt gaaggtgtcc 120 tgcaaggcct ctggctaccc cgtgaccgcc tactacatca gctggatcag acaggcccca 180 ggccagggcc tggaatggat cggctacatc gacatgggca acggccggac caactacaac 240 gcccggttta agggcagagc caccctgacc gtggacaaga gcaccagcac cgcctacatg 300 gaactgagca gcctgcggag cgaggacacc gccgtgtact actgcgccag agacagcaac 360 tggggcgtgg actattgggg ccagggcaca ctcgtgaccg tcagctcagc ctccaccaag 420 ggcccaagcg tcttccccct ggcaccctcc tccaagagca cctctggcgg cacagccgcc 480 ctgggctgcc tggtcaagga ctacttcccc gaacccgtga ccgtgagctg gaactcaggc 540 gccctgacca gcggcgtgca caccttcccc gctgtcctgc agtcctcagg actctactcc 600 ctcagcagcg tggtgaccgt gccctccagc agcttgggca cccagaccta catctgcaac 660 gtgaatcaca agcccagcaa caccaaggtg gacaagagag ttgagcccaa atcttgtgac 720 aaaactcaca catgcccacc ctgcccagca cctgaactcc tggggggacc ctcagtcttc 780 ctcttccccc caaaacccaa ggacaccctc atgatctccc ggacccctga ggtcacatgc 840 gtggtggtgg acgtgagcca cgaagaccct gaggtcaagt tcaactggta cgtggacggc 900 gtggaggtgc ataatgccaa gacaaagccc cgggaggagc agtacaacag cacgtaccgg 960 gtggtcagcg tcctcaccgt cctgcaccag gactggctta atggcaagga gtacaagtgc 1020 aaggtctcca acaaagccct cccagccccc atcgagaaaa ccatctccaa agccaaaggc 1080 cagccccggg aaccacaggt gtacaccctg cccccatccc gggaggagat gaccaagaac 1140 caggtcagcc tgacctgcct ggtcaaaggc ttctatccca gcgacatcgc cgtggagtgg 1200 gagagcaatg gccagcccga gaacaactac aagaccaccc ctcccgtgct ggactccgac 1260 ggctccttct tcctctacag caagctcacc gtggacaaga gcaggtggca gcagggcaac 1320 gtcttctcat gctccgtgat gcatgaggct ctgcacaacc actacaccca gaagagcctc 1380 tccctgtctc ccggcaaatg a 1401 <210> 114 <211> 466 <212> PRT <213> artificial <220> <223> Amino Acid Sequence of hR198_H5 <400> 114 Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala Pro Arg Trp 1 5 10 15 Val Leu Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys             20 25 30 Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Pro Val         35 40 45 Thr Ala Tyr Ile Ser Trp Ile Arg Gln Ala Pro Gly Gln Gly Leu     50 55 60 Glu Trp Ile Gly Tyr Ile Asp Met Gly Asn Gly Arg Thr Asn Tyr Asn 65 70 75 80 Ala Arg Phe Lys Gly Arg Ala Thr Leu Thr Val Asp Lys Ser Thr Ser                 85 90 95 Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val             100 105 110 Tyr Tyr Cys Ala Arg Asp Ser Asn Trp Gly Val Asp Tyr Trp Gly Gln         115 120 125 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Ser Ser Val     130 135 140 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 145 150 155 160 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser                 165 170 175 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val             180 185 190 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro         195 200 205 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys     210 215 220 Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp 225 230 235 240 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly                 245 250 255 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile             260 265 270 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu         275 280 285 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His     290 295 300 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 305 310 315 320 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys                 325 330 335 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu             340 345 350 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr         355 360 365 Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu     370 375 380 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 385 390 395 400 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val                 405 410 415 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp             420 425 430 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His         435 440 445 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro     450 455 460 Gly Lys 465 <210> 115 <211> 48 <212> PRT <213> Homo sapiens <400> 115 Ser Gly Ser Gly Phe Leu Pro Leu Lys Lys Gln Pro Gly Gln Pro Arg 1 5 10 15 Pro Thr Ser Lys Pro Pro Ala Ser Gly Ala Ala Asn Val Ser Thr             20 25 30 Ser Gly Ile Thr Pro Gly Gln Ala Ala Ile Ala Ser Thr Thr Ile         35 40 45 <210> 116 <211> 11 <212> PRT <213> Rattus rattus <400> 116 Arg Ala Ser Gln Ser Ile Ser Asn Ser Leu Ser 1 5 10

Claims (38)

Wherein the CDRH1 comprises a variable region having CDRH1, CDRH2 and CDRH3, the CDRH1 comprises an amino acid sequence shown in SEQ ID NO: 102, and the CDRH2 has an amino acid sequence represented by SEQ ID NO: Comprising the amino acid sequence represented by any one of SEQ ID NOS: 104, 106, 107, or 108, wherein said CDRH3 comprises the amino acid sequence shown by SEQ ID NO: 109 or 110; And
Wherein the light chain sequence comprises a variable region having CDRL1, CDRL2 and CDRL3, the CDRL1 comprises an amino acid sequence represented by any one of SEQ ID NO: 93, 94 or 95, and CDRL2 is SEQ ID NO: 96, 97 or 98 CDRL3 consists of the amino acid sequence shown in SEQ ID NO: 101;
Or an antigen-binding fragment thereof. The method according to claim 1,
Wherein the CDRH1 comprises a variable region having CDRH1, CDRH2 and CDRH3, the CDRH1 comprises an amino acid sequence represented by SEQ ID NO: 102, the CDRH2 comprises an amino acid sequence represented by SEQ ID NO: 106, Consisting of the amino acid sequence shown in SEQ ID NO: 110; And
Wherein the light chain sequence comprises a variable region having CDRL1, CDRL2 and CDRL3, the CDRL1 comprises an amino acid sequence represented by SEQ ID NO: 93, the CDRL2 comprises an amino acid sequence represented by SEQ ID NO: 96, Comprising an amino acid sequence represented by SEQ ID NO: 101;
Or an antigen-binding fragment thereof. The method according to claim 1,
Wherein the CDRH1 comprises a variable region having CDRH1, CDRH2 and CDRH3, the CDRH1 comprises an amino acid sequence represented by SEQ ID NO: 102, the CDRH2 comprises an amino acid sequence represented by SEQ ID NO: 106, Consisting of the amino acid sequence shown in SEQ ID NO: 110; And
Wherein the light chain sequence comprises a variable region having CDRL1, CDRL2 and CDRL3, the CDRL1 comprises an amino acid sequence shown by SEQ ID NO: 94, the CDRL2 comprises an amino acid sequence shown by SEQ ID NO: 97, Comprising an amino acid sequence represented by SEQ ID NO: 101;
Or an antigen-binding fragment thereof. The method according to claim 1,
Wherein the CDRH1 comprises a variable region having CDRH1, CDRH2 and CDRH3, the CDRH1 comprises an amino acid sequence represented by SEQ ID NO: 102, the CDRH2 comprises an amino acid sequence represented by SEQ ID NO: 106, Consisting of the amino acid sequence shown in SEQ ID NO: 110; And
Wherein the light chain sequence comprises a variable region having CDRL1, CDRL2 and CDRL3, the CDRL1 comprises the amino acid sequence shown by SEQ ID NO: 95, the CDRL2 comprises the amino acid sequence shown by SEQ ID NO: 98, Comprising an amino acid sequence represented by SEQ ID NO: 101;
Or an antigen-binding fragment thereof. The method according to claim 1,
Wherein the CDRH1 comprises a variable region having CDRH1, CDRH2 and CDRH3, the CDRH1 comprises an amino acid sequence shown by SEQ ID NO: 102, the CDRH2 comprises an amino acid sequence shown by SEQ ID NO: 107, Consisting of the amino acid sequence shown in SEQ ID NO: 110; And
Wherein the light chain sequence comprises a variable region having CDRL1, CDRL2 and CDRL3, the CDRL1 comprises an amino acid sequence represented by SEQ ID NO: 93, the CDRL2 comprises an amino acid sequence represented by SEQ ID NO: 96, Comprising an amino acid sequence represented by SEQ ID NO: 101;
Or an antigen-binding fragment thereof. The method according to claim 1,
Wherein the CDRH1 comprises a variable region having CDRH1, CDRH2 and CDRH3, the CDRH1 comprises an amino acid sequence shown by SEQ ID NO: 102, the CDRH2 comprises an amino acid sequence shown by SEQ ID NO: 108, Consisting of the amino acid sequence shown in SEQ ID NO: 110; And
Wherein the light chain sequence comprises a variable region having CDRL1, CDRL2 and CDRL3, the CDRL1 comprises an amino acid sequence represented by SEQ ID NO: 93, the CDRL2 comprises an amino acid sequence represented by SEQ ID NO: 96, Comprising an amino acid sequence represented by SEQ ID NO: 101;
Or an antigen-binding fragment thereof. The method according to claim 1,
Wherein the CDRH1 comprises a variable region having CDRH1, CDRH2 and CDRH3, the CDRH1 comprises an amino acid sequence represented by SEQ ID NO: 102, the CDRH2 comprises an amino acid sequence represented by SEQ ID NO: 104, An amino acid sequence shown by SEQ ID NO: 109; And
Wherein the light chain sequence comprises a variable region having CDRL1, CDRL2 and CDRL3, the CDRL1 comprises an amino acid sequence represented by SEQ ID NO: 93, the CDRL2 comprises an amino acid sequence represented by SEQ ID NO: 96, Comprising an amino acid sequence represented by SEQ ID NO: 101;
Or an antigen-binding fragment thereof. 3. The method according to claim 1 or 2,
A heavy chain variable region sequence consisting of amino acid residues 20 to 136 of the amino acid sequence shown in SEQ ID NO: 62 and an light chain variable region sequence composed of the amino acid residues 21 to 126 of the amino acid sequence shown in SEQ ID NO: 56 &Lt; / RTI &gt; or an antigen-binding fragment thereof. 9. The method of claim 8,
A heavy chain sequence consisting of amino acid residues 20 to 465 or 20 to 466 of the amino acid sequence shown in SEQ ID NO: 62, and a light chain sequence consisting of amino acid residues 21 to 234 of the amino acid sequence shown in SEQ ID NO: 56 &Lt; / RTI &gt; or an antigen-binding fragment thereof. 9. The method of claim 8,
A heavy chain sequence consisting of amino acid residues 20 to 465 or 20 to 466 of the amino acid sequence shown in SEQ ID NO: 70 and amino acid residues 21 to 234 of the amino acid sequence shown in SEQ ID NO: 56 &Lt; / RTI &gt; or an antigen-binding fragment thereof. The method according to claim 1 or 3,
A heavy chain variable region sequence consisting of amino acid residues 20 to 136 of the amino acid sequence shown in SEQ ID NO: 62 and an light chain variable region sequence composed of amino acid residues 21 to 126 of the amino acid sequence shown in SEQ ID NO: 58 &Lt; / RTI &gt; or an antigen-binding fragment thereof. 12. The method of claim 11,
A heavy chain sequence consisting of amino acid residues 20 to 465 or 20 to 466 of the amino acid sequence shown in SEQ ID NO: 62 and amino acid residues 21 to 234 of the amino acid sequence shown in SEQ ID NO: 58 &Lt; / RTI &gt; or an antigen-binding fragment thereof. The method according to claim 1 or 4,
A heavy chain variable region sequence consisting of amino acid residues 20 to 136 of the amino acid sequence shown in SEQ ID NO: 62 and an light chain variable region sequence consisting of the 21 to 126th amino acid residues of the amino acid sequence shown in SEQ ID NO: 60 &Lt; / RTI &gt; or an antigen-binding fragment thereof. 14. The method of claim 13,
An amino acid residue in the 20th to 465th or 20th to 466th amino acid residues of the amino acid sequence shown in SEQ ID NO: 62, and an amino acid residue in the 21st to 234th positions in the amino acid sequence shown in SEQ ID NO: 60 &Lt; / RTI &gt; or an antigen-binding fragment thereof. 6. The method according to claim 1 or 5,
A heavy chain variable region sequence consisting of amino acid residues 20 to 136 of the amino acid sequence shown by SEQ ID NO: 64 and an light chain variable region sequence consisting of amino acid residues 21 to 126 of the amino acid sequence shown by SEQ ID NO: 56 &Lt; / RTI &gt; or an antigen-binding fragment thereof. 16. The method of claim 15,
An amino acid residue in the 20th to 465th or 20th to 466th amino acid residues of the amino acid sequence shown by SEQ ID NO: 64, and an amino acid residue in the 21st to 234th positions of the amino acid sequence shown in SEQ ID NO: 56 &Lt; / RTI &gt; or an antigen-binding fragment thereof. 7. The method according to claim 1 or 6,
A heavy chain variable region sequence consisting of amino acid residues 20 to 136 of the amino acid sequence shown by SEQ ID NO: 66, and light chain variable region arrays comprising the amino acid residues 21 to 126 of the amino acid sequence shown by SEQ ID NO: 56 &Lt; / RTI &gt; or an antigen-binding fragment thereof. 18. The method of claim 17,
A heavy chain sequence consisting of the 20th to 465th or 20th to 466th amino acid residues of the amino acid sequence shown by SEQ ID NO: 66 and an 21st to 234th amino acid residue of the amino acid sequence shown by SEQ ID NO: 56 &Lt; / RTI &gt; or an antigen-binding fragment thereof. 8. The method of claim 1 or 7,
A heavy chain variable region sequence consisting of amino acid residues 20 to 136 of the amino acid sequence shown in SEQ ID NO: 43, and light chain variable region arrays consisting of amino acid residues 21 to 126 of the amino acid sequence shown in SEQ ID NO: 56 &Lt; / RTI &gt; or an antigen-binding fragment thereof. 20. The method of claim 19,
An amino acid residue in the 20th to 465th or 20th to 466th amino acid residues of the amino acid sequence shown in SEQ ID NO: 43, and an amino acid residue in the 21st to 235th positions in the amino acid sequence shown in SEQ ID NO: 56 &Lt; / RTI &gt; or an antigen-binding fragment thereof. 21. The method according to any one of claims 1 to 20,
Fab, F (ab ') 2, Fab' and Fv. The method according to any one of claims 1 to 8, 11, 13, 15, 17 or 19,
scFv. &lt; / RTI &gt; A pharmaceutical composition comprising at least one of the antibody according to any one of claims 1 to 22 or the antigen-binding fragment thereof. 24. The method of claim 23,
An anti-platelet or anti-thrombocyte activator, an Orai1-expressing cell activation inhibitor, an agent for treating or preventing an immune-related disease, an allergic disease, an inflammatory disease, a cancer. 25. The method of claim 24,
The rejection of the graft is a graft caused by rejection of organ or tissue transplantation such as heart, kidney, liver, bone marrow, skin, and host-to-graft reaction and transplantation of hematopoietic cells (bone marrow, peripheral blood, umbilical cord blood, etc.) Lt; RTI ID = 0.0 &gt; a &lt; / RTI &gt; major host disease. 25. The method of claim 24,
Immune-related diseases include, but are not limited to, connective tissue or musculoskeletal system (arthritic rheumatism, ankylosing spondylitis, systemic erythematosus, nephelineosis, dermatomyositis, dermatomyositis), blood system (aplastic anemia, idiopathic thrombocytopenic purpura, (Such as Crohn's disease and ulcerative colitis), nervous system (multiple sclerosis, myasthenia gravis, etc.), visual system (uveitis), vascular system (Behcet's disease, Wegener's granulomatosis etc.), epidermal system (psoriasis, pemphigus, (Allergic diseases, atopic dermatitis, asthma, anaphylaxis, anaphylactoid reaction, food allergy, rhinitis, otitis media, drug reaction, insect phase reaction) Inflammatory diseases such as glomerulonephritis (glomerulonephritis, nephrosis), inflammatory lung diseases (chronic obstructive pulmonary disease), chronic obstructive pulmonary disease , Inflammatory bowel disease (such as ulcerative colitis, ileitis), inflammatory liver disease (such as autoimmune hepatitis, viral hepatitis), inflammatory heart disease (myocarditis, ischemic heart disease, atherosclerosis, etc.) Inflammatory diseases such as arthritis, inflammatory diseases such as inflammatory diseases such as inflammatory skin diseases (such as contact dermatitis and eczema), inflammatory eye diseases (such as trachoma and endophthalmitis), inflammatory central nervous diseases (meningitis, Osteoarthritis, etc.), systemic inflammation (sepsis, bleeding, hypersensitivity, shock symptoms caused by cancer chemotherapy, etc.). 25. The method of claim 24,
Examples in which the treatment and / or prevention of cancer are breast cancer, lung cancer, skin cancer, leukemia, anti-platelet or anti-thrombotic activity are useful for treatment and / or prevention are myocardial infarction, stroke, ischemic heart disease, thrombosis, Examples where the inhibition of activation is helpful in the treatment and / or prevention are mast cell leukemia, mastocytosis, basophilic leukemia, endometriosis, stromalcytosis, sclerotic myopathy, arthritic rheumatism, ankylosing spondylitis, atopic dermatitis &Lt; / RTI &gt; 22. A polynucleotide encoding an antibody or antigen-binding fragment thereof of any one of claims 1 to 22. 29. A vector comprising any one of the polynucleotides of claim 28. 29. A transformed host cell comprising any one of the polynucleotides of claim 28. 29. A transformed host cell comprising the vector of claim 29. 28. A method for producing an antigen-binding fragment of an antibody or an antibody thereof according to any one of claims 1 to 22, which comprises culturing the host cell according to claim 30 or 32 and purifying the antibody from the culture product Way. Characterized in that the concentration specifically binding to the amino acid sequence set forth in SEQ ID NO: 2 and inhibiting 50% of the IL-2 released by PMA and A23187-treated Jurkat cells is 80 ng / ml or less. Antigen-binding fragment. 34. The method of claim 33,
Wherein the concentration that inhibits 50% of the amount of IL-2 released by PMA and A23187-treated Jurkat cells is 10 ng / ml or less. Characterized in that the antibody specifically binds to the amino acid sequence set forth in SEQ ID NO: 2 and has a concentration that inhibits 50% of the amount of IL-2 released by PMA and A23187-treated human peripheral blood mononuclear cells to 100 ng / Antigen-binding fragment of an antibody. 36. The method of claim 35,
Wherein the concentration that inhibits 50% of the amount of IL-2 released by PMA and A23187-treated human peripheral blood mononuclear cells is 20 ng / ml or less. Characterized in that the antibody specifically binds to the amino acid sequence set forth in SEQ ID NO: 2 and has a concentration that inhibits 50% of the amount of IFN-y released by PMA and A23187-treated human peripheral blood mononuclear cells to 800 ng / Antigen-binding fragment of an antibody. 39. The method of claim 37,
Wherein the concentration that inhibits 50% of the amount of IFN-y released by PMA and A23187-treated human peripheral blood mononuclear cells is 40 ng / ml or less.

Images